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Patent 2547511 Summary

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(12) Patent Application: (11) CA 2547511
(54) English Title: RECOMBINANT ICOSAHEDRAL VIRUS LIKE PARTICLE PRODUCTION IN PSEUDOMONADS
(54) French Title: PRODUCTION DE PARTICULES DU TYPE VIRUS ICOSAEDRIQUE RECOMBINE CHEZ DES PSEUDOMONADES
Status: Dead
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 15/83 (2006.01)
  • C12N 1/21 (2006.01)
  • C12N 15/33 (2006.01)
  • C12N 15/62 (2006.01)
  • C12N 15/63 (2006.01)
  • C12P 21/02 (2006.01)
(72) Inventors :
  • RASOCHOVA, LADA (United States of America)
  • DAO, PHILIP PHUOC (United States of America)
(73) Owners :
  • PFENEX INC. (United States of America)
(71) Applicants :
  • DOW GLOBAL TECHNOLGIES INC. (United States of America)
(74) Agent: SIM & MCBURNEY
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 2004-12-01
(87) Open to Public Inspection: 2005-07-28
Examination requested: 2009-11-18
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US2004/040117
(87) International Publication Number: WO2005/067478
(85) National Entry: 2006-05-29

(30) Application Priority Data:
Application No. Country/Territory Date
60/525,982 United States of America 2003-12-01

Abstracts

English Abstract




The present invention provides an improved process for the production of
recombinant peptides by fusion of recombinant peptides with icosahedral viral
capsids and expression of the fusion in bacterial cells of Pseudomonad origin.
The Pseudomonad cells support formation of virus like particles from
icosahedral viral capsids in vivo, and allow the inclusion of larger
recombinant peptides as monomers or concatamers in the virus like particle.
The invention specifically provides cells expressing viral capsid fusions,
nucleic acids encoding fusions of toxic proteins with icosahedral viral
capsids and processes for manufacture of recombinant proteins.


French Abstract

L'invention concerne un procédé amélioré de production de peptides recombinés par fusion de peptides recombinés avec des capsides virales icosaédriques et l'expression de la fusion chez les cellules bactériennes d'origine Pseudomonade. Les cellules de Pseudomonades supportent la formation de particules du type virus à partir de capsides virales icosaédriques in vivo et permettent l'inclusion de peptides recombinés plus grands en tant que monomères ou que concatémères dans la particule du type virus. D'une manière plus spécifique, l'invention concerne des cellules exprimant des fusions de capsides virales, des fusions de codage d'acide nucléique de protéines toxiques avec des capsides virales icosaédriques et des procédés de fabrication de protéines recombinées.

Claims

Note: Claims are shown in the official language in which they were submitted.




CLAIMS
1) A Pseudomonad cell that comprises a first nucleic acid construct
comprising:
a) at least one nucleic acid sequence encoding a icosahedral viral capsid; and
b) at least one nucleic acid sequence encoding a recombinant peptide.
2) The cell of claim 1, wherein the Pseudomonad is Pseudomonas fluorescens.
3) The cell of claim 1, wherein the icosahedral viral capsid is from a virus
that
does not display a native tropism to a Pseudomonad cell.
4) The cell of claim 3, wherein the icosahedral viral capsid is from a plant
icosahedral virus.
5) The cell of claim 4, wherein the plant icosahedral virus is selected from
the
group consisting of a Cowpea Chlorotic Mottle Virus, a Cowpea Mosaic Virus,
and an Alfalfa Mosaic Virus.
6) The cell of claim 1, wherein the nucleic acid encodes at least two
different
icosahedral viral capsids.
7) The cell of claim 6, wherein at least one of the icosahedral viral capsids
is
from a plant icosahedral virus.
8) The cell of claim 1, wherein the nucleic acid encoding the recombinant
peptide contains more than one monomer.
9) The cell of claim 8, wherein the nucleic acid encoding the recombinant
peptide contains at least three monomers.
10) The cell of claim 8, wherein the monomers are operably linked as
concatamers.
11) The cell of claim 1, wherein the recombinant peptide fused to the
icosahedral
capsid is a therapeutic peptide.
12) The cell of claim 1, wherein the recombinant peptide is an antigen.
13) The cell of claim 12, wherein the antigen is selected from the group
consisting
of a Canine Parvovirus antigen, a Bacillus Anthracis antigen, and an Eastern
Equine Encephalitis viral antigen.
14) The cell of claim 1, wherein the recombinant peptide is an antimicrobial
peptide.
15) The cell of claim 14, wherein the antimicrobial peptide is selected from
the
group consisting of D2A21 and PBF20.
78




16) The cell of claim 1, wherein the recombinant peptide is at least 7 amino
acids
in length.
17) The cell of claim 16, wherein the recombinant peptide is at least 15 amino
acids in length.
18) The cell of claim 1, wherein the cell further comprises a second nucleic
acid
encoding a wild type icosahedral viral protein.
19) The cell of claim 1, wherein the cell further comprises a second nucleic
acid
comprising:
c) at least one nucleic acid sequence encoding a second icosahedral viral
capsid; and
d) at least one nucleic acid sequence encoding a second recombinant peptide.
20) The cell of claim 19 wherein the first and second icosahedral viral
capsids are
different.
21) A Pseudomonad cell that comprises a fusion peptide, wherein the fusion
peptide comprises:
a) at least one icosahedral viral capsid; and
b) at least one recombinant peptide.
22) The cell of claim 21 wherein the fusion peptide assembles within the cell
to
form a virus like particle.
23) The cell of claim 21 wherein the fusion peptide assembles within the cell
to
form a soluble cage structure.
24) The cell of claim 22, wherein the virus like particle is not capable of
replication.
25) The cell of claim 22, wherein the virus like particle is not capable of
infecting
a cell.
26) The cell of claim 21, wherein the recombinant peptide is inserted into at
least
one surface loop of the icosahedral capsid.
27) The cell of claim 21, wherein a recombinant peptide is inserted into more
than
one surface loop of the icosahedral capsid.
28) The cell of claim 21, wherein the fusion peptide comprises more than one
recombinant peptide fused to an icosahedral viral capsid.
29) The cell of claim 28, wherein the recombinant peptides are different.
30) The cell of claim 21, wherein the recombinant peptide is a therapeutic
peptide.
31) The cell of claim 21, wherein the recombinant peptide is an antigen.
79




32) The cell of claim 31, wherein the antigen is selected from the group
consisting
of a Canine Parvovirus antigen, a Bacillus Anthracis antigen, and an Eastern
Equine Encephalitis viral antigen.
33) The cell of claim 22, wherein the virus like particle is capable of use as
a
vaccine.
34) The cell of claim 21, wherein the recombinant peptide is a peptide that is
an
antimicrobial peptide.
35) The cell of claim 34, wherein the antimicrobial peptide is selected from
the
group consisting of D2A21 and PBF20.
36) The cell of claim 21, wherein the recombinant peptide is at least 7 amino
acids
in length.
37) The cell of claim 21, wherein the recombinant peptide is at least 15 amino
acids in length.
38) The cell of claim 21, wherein the cell further comprises a wild type
icosahedral viral capsid.
39) The cell of claim 21, wherein the cell further comprises a second fusion
peptide comprising:
a) at least a second icosahedral viral capsid; and
b) at least a second recombinant peptide.
40) The cell of claim 39 wherein the second fusion peptide assembles within
the
cell to form a virus like particle or a soluble cage structure.
41) The cell of claim 39 wherein the second fusion peptide comprises a
different
amino acid sequence than the first fusion peptide.
42) The cell of claim 21 wherein the viral capsid and the recombinant peptide
are
linked by an amino acid sequence comprising a linker.
43) The cell of claim 42 wherein the linker amino acid sequence comprises a
cleavable sequence.
44) The cell of claim 21, wherein the Pseudomonad is Pseudomonas fluorescens.
45) A nucleic acid construct comprising a first nucleic acid sequence encoding
an
icosahedral viral capsid operably linked to a second nucleic acid sequence
encoding a peptide that is toxic to a microbial cell.
46) The construct of claim 45, wherein the icosahedral viral capsid is from a
plant
icosahedral virus.


47) The construct of claim 46, wherein the plant icosahedral virus is selected
from
the group consisting of a Cowpea Chlorotic Mottle Virus, a Cowpea Mosaic
Virus, and an Alfalfa Mosaic Virus.
48) The construct of claim 46, wherein the toxic peptide comprises more than
one
peptide monomer sequence.
49) The construct of claim 46, wherein the toxic peptide comprises at least
three
peptide monomer sequences.
50) The construct of claim 48, wherein the monomers are operably linked to
form
a concatamer.
51) The construct of claim 45, wherein the operable linkage is internal to the
first
nucleic acid sequence encoding the capsid.
52) The construct of claim 45 wherein the second nucleic acid sequence
encoding
the toxic peptide is operably linked to the capsid sequence in a location
encoding for at least one surface loop of the capsid.
53) The construct of claim 45, wherein the construct encodes more than one
toxic
peptide sequence operably linked to the capsid sequence locations encoding
for more than one surface loop of the capsid.
54) The construct of claim 45, wherein the recombinant peptide is an
antimicrobial
peptide.
55) The construct of claim 54, wherein the antimicrobial peptide is selected
from
the group consisting of D2A21 and PBF20.
56) A process for producing a recombinant peptide comprising:
a) providing a Pseudomonad cell;
b) providing a nucleic acid encoding a fusion peptide, wherein the fusion
peptide comprises at least one recombinant peptide and at least one
icosahedral capsid;
c) expressing the nucleic acid in the Pseudomonad cell, wherein the fusion
peptide assembles into virus like particles; and
d) isolating the virus like particles.
57) The process of claim 56, further comprising:
e) cleaving the fusion peptide to separate the recombinant peptide from the
icosahedral viral capsid.
58) The process of claim 56, wherein the Pseudomonad is Pseudomonas
fluorescens.
81


59) The process of claim 56, wherein the virus like particle is not capable of
replication.
60) The process of claim 56, wherein the virus like particle is not capable of
infecting a cell.
61) The process of claim 56, wherein the icosahedral viral capsid is from a
virus
that does not display a native tropism to a Pseudomonad cell.
62) The process of claim 56, wherein the icosahedral viral capsid is from a
plant
icosahedral virus.
63) The process of claim 62, wherein the plant icosahedral virus is selected
from
the group consisting of a Cowpea Chlorotic Mottle Virus, a Cowpea Mosaic
Virus, and an Alfalfa Mosaic Virus.
64) The process of claim 56, wherein the nucleic acid comprises a nucleic acid
sequence encoding at least two different icosahedral viral capsids.
65) The process of claim 64, wherein at least one of the icosahedral viral
capsids
is from a plant icosahedral virus.
66) The process of claim 56, wherein the recombinant peptide comprises more
than one peptide monomer.
67) The process of claim 56, wherein the recombinant peptide comprises at
least
three monomers.
68) The process of claim 66, wherein the monomers are operably linked as a
concatamer.
69) The process of claim 56, wherein the recombinant peptide is operably
linked
to at least one surface loop of the icosahedral capsid.
70) The process of claim 69, wherein a recombinant peptide is operably linked
to
more than one surface loop of the icosahedral capsid.
71) The process of claim 56, wherein the fusion peptide comprises more than
one
recombinant peptide, the recombinant peptides being dissimilar.
72) The process of claim 56, wherein the recombinant peptide is a therapeutic
peptide.
73) The process of claim 56, wherein the recombinant peptide is an antigen.
74) The process of claim 73, wherein the antigen is selected from the group
consisting of a Canine Parvovirus antigen, a Bacillus Anthracis antigen, and
an Eastern Equine Encephalitis viral antigen.
82


75) The process of claim 56, wherein the virus like particle is capable of use
as a
vaccine.
76) The process of claim 56, wherein the recombinant peptide is a peptide that
is
an antimicrobial peptide.
77) The process of claim 76, wherein the antimicrobial peptide is selected
from
the group consisting of D2A21 and PBF20.
78) The process of claim 56, wherein the recombinant peptide is at least 7
amino
acids in length.
79) The process of claim 56, wherein the recombinant peptide is at least 15
amino
acids in length.
80) The process of claim 56, wherein the cell further comprises a second
nucleic
acid encoding a wild type icosahedral viral capsid.
81) The process of claim 56, wherein the cell further comprises a second
nucleic
acid encoding a second fusion peptide comprising:
a) at least a second icosahedral viral capsid; and
b) at least a second recombinant peptide.
82) The process of claim 81 comprising expressing the second nucleic acid in
the
cell.
83) The process of claim 81 wherein the second fusion peptide assembles within
the cell to form a virus like particle or a soluble cage structure.
84) The process of claim 81 wherein the first icosahedral viral capsid
comprises a
first amino acid sequence and the second icosahedral viral capsid comprises a
second amino acid sequence and the first and second capsid sequences are
different.
85) The process of claim 81 wherein the first recombinant peptide comprises a
first amino acid sequence and the second recombinant peptide comprises a
second amino acid sequence and the first and second recombinant peptide
sequences are different.
83

Description

Note: Descriptions are shown in the official language in which they were submitted.



CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
RECOMBINANT ICOSAHEDRAL VIRUS LIKE PARTICLE
PRODUCTION IN PSEUDOMONADS
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional patent application serial
No. 60/525,982 filed December 1, 2003, entitled "High Efficiency Peptide
Production
in Pseudomonads."
FIELD OF THE INVENTION
The present invention provides an improved process for the production of
recombinant peptides. In particular, the present invention provides an
improved
process for the production or presentation of recombinant peptides in
bacterial cells
utilizing virus like particles from icosahedral viruses.
BACKGROUND OF THE INVENTION
The genetic engineering revolution has expanded to the development of
recombinant peptides for use as human and animal therapeutics. At present,
there are °
more than 100 biotechnology-derived therapeutics and vaccines approved by the
U.S.
FDA for medical use and over 1000 additional drugs and vaccines are in various
phases of clinical trials. (See M. Rai & H. Padh, (2001) "Expression systems
for
production of heterologous proteins," Cur. Science 80(9):1121-1128).
Bacterial, yeast, Dictyostelium discoideuna, insect, and mammalian cell
expression systems are currently used to produce recombinant peptides, with
varying
degrees of success. One goal in creating expression systems for the production
of
heterologous peptides is to provide broad based, flexible, efficient,
economic, and
practical platforms and processes that can be utilized in commercial,
therapeutic, and
vaccine applications. For example, for the production of certain peptides, it
would be
ideal to have an expression system capable of producing, in an efficient and
inexpensive manner, large quantities of final, desirable products in vivo in
order to
eliminate or reduce downstream reassembly costs.
1


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
Currently, bacteria are the most widely used expression system for the
production of recombinant peptides because of their potential to produce
abundant
quantities of recombinant peptides. However, bacteria are often limited in
their
capacities to produce certain types of peptides, requiring the use of
alternative, and
more expensive, expression systems. For example, bacteria systems are
restricted in
their capacity to produce monomeric antimicrobial peptides due to the toxicity
of such
peptides to the bacteria, often leading to the death of the cell upon the
expression of
the peptide. Because of the inherent disadvantages in terms of the costs and
product
yields of non-bacterial expression systems, significant time and resources
have been
spent on trying to improve the capacity of bacterial systems to produce a wide
range
of commercially and therapeutically useful peptides. While progress has been
made
in this area, additional processes and platforms for the production of
heterologous
peptides in bacterial expression systems would be beneficial.
Yi~uses
One approach for improving peptide production in host cell expression
systems is to make use of the properties of replicative viruses to produce
recombinant
peptides of interest. However, the use of replicative, full length viruses has
numerous
drawbaclcs for use in recombinant peptide production strategies. For example,
it may
be difficult to control recombinant peptide production during fermentation
conditions,
which may require tight regulation of expression in order to maximize
efficiency of
the fermentation run. Furthermore, the use of replicative viruses to produce
recombinant peptides may result in the imposition of regulatory requirements,
which
may lead to increased downstream purification steps.
To overcome production issues particularly during fermentation, one area of
research has focused on the expression and assembly of viruses in a cell that
is not a
natural host to the particular virus (a non-tropic host cell). A non-tropic
cell is a cell
that the virus is incapable of successfully entering due to incompatibility
between
virus capsids and the host receptor molecules, or an incompatibility between
the
biochemistry of the virus and the biochemistry of the cell, preventing the
virus from
completing its life cycle. For example, US Patent No. 5,869,287 to Price et
al.
describes a method for synthesizing and assembling, in yeast cells, replicable
or
infectious viruses containing RNA, where either the viral capsids or the RNA
contained within the capsids are from a non-yeast virus species of the
Nodaviridae or
2


CA 02547511 2006-05-29
WO 2005/067478 . PCT/US2004/040117
Bromoviridae. However, this approach does not overcome the potential
regulatory
hurdles that are associated with protein production in replicative viruses.
Tlirus Like Particles
Another approach for improving the production of recombinant peptides has
been to use virus like particles (VLPs). In general, encapsidated viruses
include a
protein coat or "capsid" that is assembled to contain the viral nucleic acid.
Many
viruses have capsids that can be "self assembled" from the individually
expressed
capsids, both within the cell the capsid is expressed in ("iu vivo assembly")
forming
VLPs, and outside of the cell after isolation and purification ("in vitf~o
assembly").
Ideally, capsids are modified to contain a target recombinant peptide,
generating a
recombinant viral capsid-peptide fusion. The fusion peptide can then be
expressed in
a cell, and, ideally, assembled ih vivo to form recombinant viral or virus-
like particles.
This approach has been met with varying success. See, for example, C
Marusic et al., J T~i~ol. 75(18):8434-39 (Sep 2001) (expression in plants of
recombinant, helical potato virus X capsids terminally fused to an antigenic,
HIV
peptide, with iu vivo formation of recombinant virus particles); FR Brennan et
al.,
haccine 17(15-16):1846-57 (09 Apr 1999) (expression in plants of recombinant,
icosahedral cowpea mosaic virus or helical potato virus X capsids terminally
fused to
an antigenic, Staphylococcus au~eus peptide, with ifa vivo formation of
recombinant
virus particles).
US Patent No. 5,874,087 to Lomonossoff & Johnson describes production of
recombinant plant viruses, in plant cells, where the viral capsids include
capsids
engineered to contain a biologically active peptide, such as a hormone, growth
factor,
or antigenic peptide. A virus selected from the genera Comovi~us,
Toynbusvirus,
Sobenaovif°us, and Nepovirus is engineered to contain the exogenous
peptide encoding
sequence and the entire engineered genome of the virus is expressed to produce
the
recombinant virus. The exogenous peptide-encoding sequence is inserted within
one
or more of the capsid surface loop motif encoding sequences.
Attempts have been made to utilize non-tropic cells to produce particular
virus
like particles. See, for example, JW Lamb et al., J Gen. Virol. 77(Pt.7):1349-
58 (Jul
1996), describing expression in insect cells of recombinant, icosahedral
potato leaf
roll virus capsids terminally fused to a heptadecapeptide, with ira vivo
formation of
virus-like particles. In certain situations, a non-tropic VLP may be
preferable. For
3


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
instance, a non-tropic viral capsid may be more accommodating to foreign
peptide
insertion without disrupting the ability to assemble into virus like particles
than a
native viral capsid. Alternatively, the non-tropic viral capsid may be better
characterized and understood than a capsid from a native, tropic virus. In
addition,
the particular application, such as vaccine production, may not allow for the
use of a
tropic virus in a particular host cell expression system. U.S. Patent No.
6,232,099 to
Chapman et al. describes the use of rod-shaped viruses to produce foreign
proteins
connected to viral capsid subunits in plants. Rod-shaped viruses, also
classified as
helical viruses, such as potato virus X (PVX) have recombinant peptides of
interest
inserted into the genome of the virus to create recombinant viral capsid-
peptide
fusions. The recombinant virus is then used to infect a host cell, and the
virus actively
replicates in the host cell and further infects other cells. Ultimately, the
recombinant
viral capsid-peptide fusion is purified from the plant host cells.
Use of Virus Like Particles ira Bacterial Expression Systems
Because of the potential of fast, efficient, inexpensive, and abundant yields
of
recombinant peptides, bacteria have been examined as host cells in expression
systems for the production of recombinant viral capsid-peptide fusion viral
like
particles.
Researchers have shown that particular wild-type viral capsids without
recombinant peptide inserts can be transgenically expressed in non-tropic
enterobacteria. Researchers have also shown that these capsids can be
assembled,
both ira vivo and in vitro, to form virus like particles. See, for example, SJ
Shire et al.,
Biochemistry 29(21):5119-26 (29 May 1990) (ifz vitro assembly of virus-like
particles
from helical tobacco mosaic virus capsids expressed in E. coli); X Zhao et
al.,
virology 207(2):486-94 (10 Mar 1995) (in vitro assembly of virus-like
particles from
icosahedral cowpea chlorotic mottle virus capsids expressed in E. coli); Y
Stram et al.,
Virus Res. 28(1):29-35 (Apr 1993) (expression of filamentous potato virus Y
capsids
in E. coli, with ira vivo formation of virus-like particles); J Joseph & HS
Savithri, AYCIa.
Yif°ol. 144(9):1679-87 (1999) (expression of filamentous chili pepper
vein banding
virus capsids in E. coli, with in vivo formation of virus-like particles); DJ
Hwang et
al., Proc. Nat'l Acad. Sci. USA 91(19):9067-71 (13 Sep 1994) (expression of
helical
tobacco mosaic virus capsids in E. coli, with in vivo formation of virus-like
particles);
4


CA 02547511 2006-05-29
WO 2005/067478 - PCT/US2004/040117
M Sastri et al., J Mol. Biol. 272(4):541-52 (03 Oct 1997) (expression of
icosahedral
physalis mottle virus capsids in E. coli, with in vivo formation of virus-like
particles).
To date, successful expression and irz vivo assembly of recombinant viral
capsid-peptide fusion particles in a non-tropic bacterial cell has been
varied. In
general, successful izz vivo assembly of these particles has been limited to
non
icosahedral virus capsid target peptide fusion particles. See, for example, MN
Jagadish et al., Ifztervi~ology 39(1-2):85-92 (1996) (expression in non-plant
cells of
recombinant, filamentous, non-icosahedral Johnsongrass mosaic virus capsids
terminally fused to an antigenic peptide, with in vivo formation of virus-like
particles).
The expression of peptides linked to icosahedral capsids has been unsuccessful
or of limited utility. For example, V Yusibov et al., J Gen. Virol.
77(Pt.4):567-73
(Apr 1996) described i>z vitro assembly of virus-like particles from E. coli-
expressed,
recombinant, icosahedral alfalfa mosaic virus capsids terminally fused to a
hexahistidine peptide.
Brumfield et al. unsuccessfully attempted to express as in vivo assembled
virus
like particles recombinant peptides inserted into an icosahedral capsid. See
Brumfield
et al., (2004) "Heterologous expression of the modified capsid of Cowpea
clzlor-otic
mottle bromovif~us results in the assembly of protein cages with altered
architectures
and functions," J. Gen. Vir. 85: 1049-1053. The reasons for the observed
inability of
icosahedral viral capsid-peptide fusion particles to assemble as virus like
particles izz
vivo in E. coli are not well understood. Brumfield at al, associate the
failure to
assemble to the fact that E. coli produces an insoluble capsid.
Chapman, in U.S. Pat. No. 6,232,099, points out that a limited insertion size
is
tolerated by icosahedral viruses. Chapman cites WO 92/18618, which limits the
size
of the recombinant peptide in an icosahedral virus for expression in a plant
host cell to
26 amino acids in length, in supporting his assertion. Chapman theorizes that
a larger
peptide present in the internal insertion site in the capsid of icosahedral
viruses may
result in disruption of the geometry of the protein and/or its ability to
successfully
interact with other capsids leading to failure of the chimeric virus to
assemble. This
reference also describes the use of non-replicative rod-shaped viruses to
produce
capsid-recombinant peptide fusion peptides in cells that can include E. coli.
Therefore, it is an object of the present invention to provide an improved
bacterial expression system for the production of virus like particles,
wherein the
virus like particle is derived from an icosahedral virus.
5


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
It is another object of the present invention to provide bacterial organisms
for
use as host cells in an improved expression system for the production of virus
like
particles.
It is still another object of the present invention to provide processes for
the
improved production of virus like particles in bacteria.
It is yet another object of the present invention to provide novel constructs
and
nucleic acids for use in an improved bacterial expression system for the
production of
virus like particles.
SUMMARY OF THE INVENTION
Icosahedral capsid-recombinant peptide fusion particles assemble into viral
like particles or soluble cage structures in vivo when expressed in
Pseudomonad
organisms. Furthermore, large recombinant peptides or peptide concatamers,
greater
than 50 amino acids, can be inserted into an icosahedral capsid and assembled
in vivo
in Pseudomonad organisms.
In one aspect of the present invention, Pseudomonad organisms are provided
that include a nucleic acid construct encoding a fusion peptide of an
icosahedral
capsid and a recombinant peptide. In one specific embodiment of the present
invention, the Pseudomonad cell is Pseudomonas fluo~~escens. In one embodiment
the
cell produces virus like particles or soluble cage structures.
The virus like particles produced in the cell typically are not capable of
infecting the cell. The viral capsid sequence can be derived from a virus not
tropic to
the cell. In one embodiment, the cell does not include viral proteins other
than the
desired icosahedral capsid. In one embodiment, the viral capsid is derived
from a
virus with a tropism to a different family of organisms than the cell. In
another
embodiment, the viral capsid is derived from a virus with a tropism to a
different
genus of organisms than the cell. In another embodiment, the viral capsid is
derived
from a virus with a tropism to a different species of organisms than the cell.
In one
embodiment of the present invention, the icosahedral capsid is derived from a
plant
icosahedral virus. In a particular embodiment, the icosahedral capsid is
derived from
the group selected from Cowpea Mosaic Virus, Cowpea Chlorotic Mottle Virus,
and
Alfalfa Mosaic Virus.
In one embodiment of the present invention, the recombinant peptide fused to
the icosahedral capsid is a therapeutic peptide useful for human or animal
treatments.
6


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
In one particular embodiment, the recombinant peptide is an antigen. In one
embodiment, the capsid-recombinant peptide virus like particles can be
administered
as a vaccine in a human or animal application. In another particular
embodiment, the
recombinant peptide is a peptide that is toxic to the host cell when in free
monomeric
form. In a more particular embodiment, the toxic peptide is an antimicrobial
peptide.
In one embodiment, the recombinant peptide fused to the icosahedral capsid is
at least 7, at least 8, at least, 9, at least 10, at least 12, at least 15, at
least 20, at least
25, at least 30, at least 35, at least 40, at least 45, at least 50, at least
55, at least 60, at
least 65, at least 75, at least 85, at least 95, at least 99, or at least 100
amino acids.
In one embodiment of the present invention, the recombinant peptide fused to
the icosahedral capsid contains at least one monomer of a desired target
peptide. In
an alternative embodiment, the recombinant peptide contains more than one
monomer
of a desired target peptide. In certain embodiments, the peptide is composed
of at
least two, at least 5, at least 10, at least 15 or at least 20 separate
monomers that are
operably linked as a concatameric peptide to the capsid. In another
embodiment, the
individual monomers in the concatameric peptide are linked by cleavable linker
regions. In still another embodiment, the recombinant peptide is inserted into
at least
one surface loop of the icosahedral viral capsid. In one embodiment, at least
one
monomer is inserted into more than one surface loops of the icosahedral viral
capsid.
More than one loop of the virus like particle can be modified. In one
particular embodiment, the recombinant peptide is expressed on at least two
surface
loops of the icosahedal virus-like particle. In another embodiment, at least
two
different peptides are inserted into at least two surface loops of the viral
capsid, cage
or virus-like particle. In another embodiment, at least three recombinant
peptides are
inserted into at least three surface loops of the virus-like particle. The
recombinant
peptides in the surface loops can have the same amino acid sequence. In
separate
embodiments, the amino acid sequence of the recombinant peptides in the
surface
loops differ.
In still another embodiment, the cell includes at least one additional nucleic
acid encoding a second either wild-type capsid or capsid-recombinant peptide
fusion
peptide, wherein the multiple capsids are assembled in vivo to produce
chimeric virus
like particles.
In one aspect of the present invention, Pseudomonad organisms are provided
that include a fusion peptide of an icosahedral capsid and a recombinant
peptide. In
7


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one specific embodiment of the present invention, the Pseudomonad cell is
Pseudomofzas fluoYescens. In one embodiment the capsid-recombinant peptide
fusion
peptide assembles in vivo to form a virus like particle.
In another aspect of the present invention, nucleic acid constructs are
provided
encoding a fusion peptide of an icosahedral capsid and a recombinant peptide.
In one
embodiment of the present invention, the icosahedral capsid is derived from a
plant
icosahedral virus. In a particular embodiment, the icosahedral capsid is
derived from
the group selected from Cowpea Mosaic Virus, Cowpea Chlorotic Mottle Virus,
and
Alfalfa Mosaic Virus.
In one embodiment, the recombinant peptide is a peptide that is toxic to the
host cell when in free monomeric form. In a more particular embodiment, the
toxic
peptide is an antimicrobial peptide.
In one embodiment of the present invention, the recombinant peptide contains
at least one monomer of a desired target peptide. In an alternative
embodiment, the
recombinant peptide contains more than one monomer of a desired target
peptide. In
still another embodiment, the recombinant peptide is inserted into at least
one surface
loop of the icosahedral virus capsid.
In another embodiment, the nucleic acid construct can include additional
nucleic acid sequences including at least one promoter, at least one selection
marker,
at least one operator sequence, at least one origin of replication, and at
least one
ribosome binding site.
In one aspect, the present invention provides a process for producing a
recombinant peptide including:
a) providing a Pseudomonad cell;
b) providing a nucleic acid encoding a fusion peptide, wherein the
fusion is of a recombinant peptide and an icosahedral capsid;
c) expressing the nucleic acid in the Pseudomonad cell, wherein the
expression in the cell provides for in vivo assembly of the fusion
peptide into virus like particles; and
d) isolating the virus like particles.
In one embodiment, the process further includes: e) cleaving the fusion
product to separate the recombinant peptide from the capsid. In one embodiment
of
the present invention, the Pseudomonad cell is Pseudonaonas fluo~escens.
8


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In one embodiment, the process includes co-expressing another nucleic acid
encoding a wild-type capsid or capsid-recombinant peptide fusion peptide,
wherein
the capsids are assembled in vivo to produce chimeric virus like particles.
In another aspect of the present invention, an expression system for the
production of recombinant peptides is provided including:
a) a Pseudomonad cell;
b) a nucleic acid encoding a fusion peptide; wherein the fusion peptide
comprises at least one recombinant peptide, and at least one
icosahedral viral capsid; and
c) a growth medium.
When expressed the fusion peptide can assemble into virus like particles
within the cell.
Brief Description of the Figures
Figure 1 presents a plasmid map of a CCMV129-CP expression plasmid useful for
expression of recombinant VLPs in Pseudomonad host cells.
Figure 2 illustrates a scheme for production of peptide monomers in Virus-Like
Particles (VLP) in host cells, e.g., Pseudomonad host cells. A desired target
peptide
insert coding sequence ("1") is inserted, in-frame, into the viral capsid
coding
sequence ("CP") in constructing a recombinant viral capsid gene ("rCP"),
which, as
part of a vector, is transformed into the host cell and expressed to form
recombinant
capsids ("rCP"). These are then assembled to form VLPs containing up to 1 ~0
rCPs
each, in the case of CCMV. The VLPs are illustrated with target peptide
inserts ("I")
expressed in external loops) of the capsid. The assembled VLPs each contain
multiple peptide inserts per particle, e.g., up to 1 ~0 or a multiple thereof.
The VLPs
are then readily precipitated from cell lysate for recovery, e.g., by PEG
precipitation.
The recombinant peptide inserts expressed in the capsid surface loops and/or
termini
can be isolated in highly pure form from the precipitated VLPs.
Figure 3 illustrates a scheme for production of peptide multimers in VLPs in
host
cells, e.g., Pseudomonad host ells. The peptide insert is a multimer (a trimer
is shown)
of the desired target peptide(s), whose coding sequences ("i") are inserted
into the
9


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viral capsid coding sequence ("CP") in constructing a recombinant viral capsid
gene
("rCP"). Each of the target peptide coding sequences is bounded by coding
sequences
for cleavage sites ("*") and the entire nucleic acid insert is labeled "1." In
the
illustration, only one trimer insertion is made per CCMV capsid, and each of
the
resulting VLPs contains up to 180 peptide inserts ("I") for a total of up to
540 target
peptides ("i"). The target peptides are then readily isolated in highly pure
form, after
precipitation of the VLPs, by treatment of the VLPs with a cleavage agent,
e.g., an
acid or an enzyme.
Figure 4 is a plasmid Map of CCMV63-CP expression plasmid useful for
expression
of recombinant VLPs. Restriction sites AscI and NotI were engineered onto CCMV-

CP (SEQ ~ NO:1) to serve as an insertion site for peptides.
Figure 5 is a plasmid Map of R26C-CCMV631129-CP expression plasmid useful for
expression of recombinant VLPs. Two insertion sites (AscI-NotI and BamHI) were
engineered in the CP for insertions of two identical or different peptides.
Figure 6 is an image of a SDS-PAGE gel showing expression of chimeric CCMV CP
in Pseudomonas fluo~escens 24 hours post induction. Chimeric CP has been
engineered to express a 20 amino acid antigenic peptide PD1. The chimeric CP
has
slower mobility compared to the non-engineered wild type (wt) CCMV CP. Lane 1
is
a size ladder, lane 2 is wild-type CP 0 hours post-induction, lane 3 is wild-
type CP 24
hours post-induction, lane 4 is CCMV 129-PD 1 0 hours post induction and lane
5 is
CCMV129-PDl 24 hours post induction.
Figure 7 is an image of a western blot showing expression of chimeric CCMV CP
in
Pseudomonas fluorescens. Chimeric CP has been engineered to express a 20 amino
acid antigenic peptide PD1. The chimeric CP has slower mobility compared to
the
non-engineered wild type (wt) CCMV CP. Lane 1 is a size ladder, lane 2 is wild-
type
CP 0 hours post-induction, lane 3 is wild-type CP 24 hours post-induction,
lane 4 is
CCMV129-PD1 0 hours post induction and lane 5 is CCMV129-PD1 24 hours post
induction.


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
Figure 8 is an image of a western blot of CCMV129-PDl VLP sucrose gradient
fractions. Chimeric CCMV CPs engineered to express a 20 amino acid antigenic
peptide PDlwere expressed in Pseudomoraas fluorescens. Chimeric VLPs were
isolated 24 hours post induction by PEG precipitation and fractionated on
sucrose
density gradient. The VLP fractions were positive for chimeric CP. Lane 1 is a
CCMV129-PD1 VLP sucrose gradient fraction, lane 2 is a CCMV129-PD1 VLP
sucrose gradient fraction, lane 3 is a CCMV129-PDl VLP sucrose gradient
fraction
and lane 4 is a size ladder.
Figure 9 is an electron microscopy (EM) image of chimeric CCMV VLPs displaying
amino acid antigenic peptides PDl. The VLPs were isolated from P. fluo~escens
using PEG precipitation and sucrose density fractionation.
Figure 10 is an image of a SDS-PAGE gel showing expression of chimeric CCMV
15 CP in Pseudomonas fluoYescens 12, 24, and 48 hours post induction. Chimeric
CP
has been engineered to express an antimicrobial peptide D2A21 trimer separated
by
acid hydrolysis sites. The chimeric CP has slower mobility compared to the non-

engineered wild type (wt) CCMV CP. Lane 1 is a size ladder, lane 2 is wild-
type CP 0
hours post induction, lane 3 is wild-type CP 12 hours post induction, lane 4
is wild-
20 type CP 24 hours post induction, lane 5 is wild-type CP 48 hours post
induction, lane
6 is CCMV129-(DZA21)3 0 hours post induction, lane 7 is CCMV129-(D2A21)3 12
hours post induction, lane 8 is CCMV129-(D2A21)3 24 hours post induction and
lane
9 is CCMV129-(D2A21)3 48 hours post induction.
Figure 11 is an image of a western blot of CCMV129-(D2A21)3 VLP sucrose
gradient fractions. Chimeric CCMV CPs engineered to express a 96 amino acid
antimicrobial peptide D2A21 trimer separated by acid hydrolysis sites were
expressed
in Pseudomonas fluoy~escens. Chimeric VLPs were isolated 24 hours post
induction by
PEG precipitation and fractionated on sucrose density gradient. The VLP
fractions
were positive for chimeric CP. Lane 1 is a size ladder, lane 2-4 are CCMV129--
(D2A21)3 VLP sucrose gradient fractions.
Figure 12 is an electron microscopy (EM) image of chimeric CCMV VLPs
displaying an antimicrobial peptide D2A21 trimer separated by acid hydrolysis
sites.
11


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The VLPs were isolated from P. fluorescens using PEG precipitation and sucrose
density fractionation.
Figure 13 is a HPLC chromatogram showing release of AMP D2A21 peptide
monomers from chimeric VLPs engineered to display an antimicrobial peptide
D2A21 trimer separated by acid cleavage sites by treatment with acid. The AMP
peptide peak has not been detected in non-engineered (empty) VLPs.
Figure 14 is a MALDI-MS graph showing the identity of AMP D2A21 peptide
monomers released from chimeric VLPs engineered to display an antimicrobial
peptide D2A21 trimer separated by acid cleavage sites by treatment with acid.
The
molecular weight is as predicted for the D2A21 peptide monomer.
Figure 15 is an image of a SDS-PAGE gel showing expression of chimeric CCMV
CP in Pseudomonas fZuorescens 12 and 24 hours post induction. Chimeric CP has
been engineered to express four different 25 amino acid antigenic peptides
PAl, PA2,
PA3, and PA4. The chimeric CP has slower mobility compared to the non-
engineered
wild type (wt) CCMV CP. Lane 1 is a size ladder, lane 2 is CCMV129-PA1 0 hours
post induction, lane 3 is CCMV129-PA1 12 hours post induction, lane 4 is
CCMV129-PAl 24 hours post induction, lane 5 is CCMV129-PA2 0 hours post
induction, lane 6 is CCMV 129-PA2 12 hours post induction, lane 7 is CCMV 129-
PA2 24 hours post induction, lane 8 is CCMV129-PA3 0 hours post induction,
lane 9
is CCMV129-PA3 12 hours post induction, lane 10 is CCMV129-PA3 24 hours post
induction, lane 11 is CCMV 129-PA4 0 hours post induction, lane 12 is CCMV 129-

PA4 12 hours post induction, lane 13 is CCMV129-PA4 24 hours post induction.
Figure 16 is an image of a western blot of CCMV129-PA1, CCMV129-PA2,
CCMV129-PA3, CCMV129-PA4 VLP sucrose gradient fractions. Chimeric CCMV
CPs engineered to express a 25 amino acid antigenic PA peptides were expressed
in
Pseudornonas fZuorescens. Chimeric VLPs were isolated 24 hours post induction
by
PEG precipitation and fractionated on sucrose density gradient. The VLP
fractions
were positive for chimeric CP. Lane 1 is a size ladder, lane 2-4 are CCMV129-
PAl
VLP sucrose gradient fractions, lanes 5-7 are CCMV129-PA2 VLP sucrose gradient
12


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
fractions, lanes 8-10 are CCMV 129-PA3 VLP sucrose gradient fractions and
lanes
11-13 are CCMV129-PA4 VLP sucrose gradient fractions.
Figure 17 is an image of a SDS-PAGE showing expression of chimeric CCMV CP in
Pseudomonas , fluo~escefzs. Chimeric CCMV63-CP has been engineered to express
a
20 amino acid antimicrobial peptide PBF20 separated by acid hydrolysis sites.
The
chimeric CP has slower mobility compared to the non-engineered wild type (wt)
CCMV CP. Lane 1 is a size ladder, lane 2 is wild-type CP 0 hours post
induction,
lane 3 is wild-type CP 24 hours post induction, lane 4 is CCMV63-PBF20 0 hours
post induction, lane 5 is CCMV63-PBF20 24 hours post induction.
Figure 18 is an electron microscopy (EM) image of chimeric CCMV VLPs derived
from CCMV63-CP and displaying a 20 amino acid antimicrobial peptide PBF20
separated by acid hydrolysis sites. The chimeric VLPs were isolated from P.
, fluorescens using PEG precipitation and sucrose density fractionation.
Figure 19 is an image of a SDS-PAGE showing expression of chimeric CCMV CP in
Pseudomonas fluo~esceus. Chimeric CCMV129-CP has been engineered to express a
amino acid antimicrobial peptide PBF20 separated by acid hydrolysis sites.
Lane 1
20 is a size ladder, lane 2 is CCMV129-PBF20 0 hours post induction and lane 3
is
CCMV129-PBF20 24 hours post induction.
Figure 20 is an electron microscopy (EM) image of chimeric CCMV VLPs derived
from CCMV 129-CP and displaying a 20 amino acid antimicrobial peptide PBF20
separated by acid hydrolysis sites. The chimeric VLPs were isolated from P.
fZuorescefzs using PEG precipitation and sucrose density fractionation.
Figure 21 is an image of a SDS-PAGE showing expression of chimeric CCMV CP in
Pseudomonas fluo~escens. Chimeric CCMV63/129-CP has been engineered to
express a 20 amino acid antimicrobial peptide PBF20 separated by acid
hydrolysis
sites in two different insertion sites in the CP (63 and 129). Chimeric CP
containing a
double insert (CP + 2x20 AA) has slower mobility on the SDS-PAGE gel compared
to the capsid engineered to express a single insert (CP + 1x20 AA) of the same
peptide. Lane 1 is a size ladder, lane 2 is CCMV63-PBF20 0 hours post
induction,
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CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
lane 3 is CCMV63-PBF20 24 hours post induction, lane 4 is CCMV63/129-
2x(PBF20) 0 hours post induction, lane 5 is CCMV63/129- 2x(PBF20) 24 hours
post
induction, lane 6 is CCMV63/129- 2x(PBF20) 0 hours post induction, lane 7 is
CCMV63/129- 2x(PBF20) 24 hours post induction, lane S is CCMV63h29-
2x(PBF20) 0 hours post induction, lane 9 is CCMV63/129- 2x(PBF20) 24 hours
post
induction.
Figure 22 is an electron microscopy (EM) image of chimeric CCMV VLPs derived
from CCMV63/129-CP displaying a 20 amino acid antimicrobial peptide PBF20
separated by acid hydrolysis sites in two insertion sites per capsid (63 and
129). The
chimeric VLPs were isolated from P. fluof-escehs using PEG precipitation and
sucrose
density fractionation.
DETAILED DESCRIPTION
The present invention provides a process for the expression in bacteria of
fusion peptides comprising an icosahedral viral capsid and a recombinant
peptide of
interest. The term "peptide" as used herein is not limited to any particular
molecular
weight, and can also include proteins or polypeptides. The present invention
further
provides bacterial cells and nucleic acid constructs for use in the process.
Specifically,
the invention provides Pseudomonad organisms with nucleic acid construct
encoding
a fusion peptide of an icosahedral capsid and a recombinant peptide. In one
specific
embodiment of the present invention, the Pseudomonad cell is Pseudomouas
fluorescens. In one embodiment the cell produces virus like particles or
soluble cage
structures. The invention also provides nucleic acid constructs encoding the
fusion
peptide of an icosahedral capsid and a recombinant peptide, which can in one
embodiment, be a therapeutic peptide useful for human and animal treatments.
The invention also provides a process for producing a recombinant peptide in
a Pseudomonad cell by providing: a nucleic acid encoding a fusion peptide of a
recombinant peptide and an icosahedral capsid; expressing the nucleic acid in
the
Pseudomonad cell, wherein the expression in the cell provides for ih vivo
assembly of
the fusion peptide into virus like particles; and isolating the virus like
particles.
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I. RECOMBINANT PSEUDOMONAD CELLS
The present invention provides Pseudomonad cells that include a nucleic acid
construct encoding a fusion peptide of an icosahedral capsid and a recombinant
peptide. The cells can be utilized in a process for producing recombinant
peptides.
Viral Capsids
In one embodiment, the invention provides Pseudomonad cells for use in a
process for producing peptides by expression of the peptide fused to an
icosahedral
viral capsid. The expression typically results in at least one virus like
particle (VLP)
in the cell.
Viruses can be classified into those with helical symmetry or icosahedral
symmetry. Generally recognized capsid morphologies include: icosahedral
(including
icosahedral proper, isometric, quasi-isometric, and geminate or "twinned"),
polyhedral (including spherical, ovoid, and lemon-shaped), bacilliform
(including
rhabdo- or bullet-shaped, and fusiform or cigar-shaped), and helical
(including rod,
cylindrical, and filamentous); any of which may be tailed and/or may contain
surface
projections, such as spikes or knobs.
Morplaology
In one embodiment of the invention, the amino acid sequence of the capsid is
selected from the capsids of viruses classified as having any icosahedral
morphology.
In one embodiment, the capsid amino acid sequence will be selected from the
capsids
of entities that are icosahedral proper. In another embodiment, the capsid
amino acid
sequence will be selected from the capsids of icosahedral viruses. In one
particular
embodiment, the capsid amino acid sequence will be selected from the capsids
of
icosahedral plant viruses. However, in another embodiment, the viral capsid
will be
derived from an icosahedral virus not infectious to plants. For example, in
one
embodiment, the virus is a virus infectious to mammals.
Generally, viral capsids of icosahedral viruses are composed of numerous
protein sub-units arranged in icosahedral (cubic) symmetry. Native icosahedral
capsids can be built up, for example, with 3 subunits forming each triangular
face of a
capsid, resulting in 60 subunits forming a complete capsid. Representative of
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CA 02547511 2006-05-29
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small viral structure is e.g. bacteriophage Q~X174. Many icosahedral virus
capsids
contain more than 60 subunits. Many capsids of icosahedral viruses contain an
antiparallel, eight-stranded beta-barrel folding motif. The motif has a wedge-
shaped
block with four beta strands (designated BIDG) on one side and four
(designated
CHEF) on. the other. There are also two conserved alpha-helices (designated A
and B),
one is between betaC and betaD, the other between betaE and betaF.
Enveloped viruses can exit an infected cell without its total destruction by
extrusion (budding) of the particle through the membrane, during which the
particle
becomes coated in a lipid envelope derived from the cell membrane (See, e.g.:
AJ
Cann (ed.) (2001) Prizzciples ofMolecula>" Tlirology (Academic Press); A
Granoff and
RG Webster (eds.) (1999) Encyclopedia of hirology (Academic Press); DLD Caspar
(1980) Bioplzys. J. 32:103; DLD Caspar and A I~lug (1962) Cold Spring HarboY
Synzp.
Quant. Biol. 27:1; J Grimes et al. (1988) Nature 395:470; JE Johnson (1996)
Proc.
Nat'l Acad. Sci. USA 93:27; and J Johnson and J Speir (1997) J. Mol. Biol.
269:665).
Tliruses
Viral taxonomies recognize the following taxa of encapsidated-particle
entities:
~ Group I Viruses, i.e. the dsDNA viruses;
~ Group II Viruses, i.e. the ssDNA viruses;
~ Group III Viruses, i.e. the dsRNA viruses;
~ Group IV Viruses, i.e. the ssRNA (+)-stranded viruses with no DNA stage;
~ Group V Viruses, i. e. the ssRNA (-)-stranded viruses;
~ Group VI Viruses, i.e. the RNA retroid viruses, which are ssRNA reverse
transcribing viruses;
~ Group VII Viruses, i.e. the DNA retroid viruses, which are dsDNA reverse
transcribing viruses;
~ Deltaviruses;
~ Viroids; and
~ Satellite phages and Satellite viruses, excluding Satellite nucleic acids
and
Prions.
Members of these taxa are well known to one of ordinary skill in the art and
are reviewed in: H.V. Van Regenmortel et al. (eds.), Virus Taxonomy: Seventh
Report of the International Committee on Taxonomy of Viruses (2000) (Academic
16


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
Press/Elsevier, Burlington Mass., USA); the Virus Taxonomy web-page of the
University of Leicester (UK) Microbiology & Immunology Department at
http://wwwmicro.msb.le.ac.uk/3035/ Virusgroups.html; and the on-line "Virus"
and
"Viroid" sections of the Taxonomy Browser of the National Center for
Biotechnology
Information (NCBI) of the National Library of Medicine of the National
Institutes of
Health of the US Department of Health ~ Human Services (Washington, D.C., USA)
at http://www.ncbi.nlm.nih.~ov/Taxonomy/ tax.html.
The amino acid sequence of the capsid may be selected from the capsids of
any members of any of these taxa. Amino acid sequences for capsids of the
members
of these taxa may be obtained from sources, including, but not limited to,
e.g.: the on-
line "Nucleotide" (Genbank), "Protein," and "Structure" sections of the PubMed
search facility offered by the NCBI at http://www.ncbi.nlm.nih.~ov/entrez/
query.fcgi.
In one embodiment, the capsid amino acid sequence will be selected from taxa
members that are specific for at least one of the following hosts: fungi
including
yeasts, plants, protists including algae, invertebrate animals, vertebrate
animals, and
humans. In one embodiment, the capsid amino acid sequence will be selected
from
members of any one of the following taxa: Group I, Group II, Group III, Group
IV,
Group V, Group VII, Viroids, and Satellite Viruses. In one embodiment, the
capsid
amino acid sequence will be selected from members of any one of these seven
taxa
that are specific for at least one of the six above-described host types. In a
more
specific embodiment, the capsid amino acid sequence will be selected from
members
of any one of Group II, Group III, Group IV, Group VII, and Satellite Viruses;
or
from any one of Group II, Group IV, Group VII, and Satellite Viruses. In
another
embodiment, the viral capsid is selected from Group IV or Group VII.
The viral capsid sequence can be derived from a virus not tropic to the cell.
In
one embodiment, the cell does not include viral proteins from the particular
selected
virus other than the desired icosahedral capsids. In one embodiment, the viral
capsid
is derived from a virus with a tropism to a different family of organisms than
the cell.
In another embodiment, the viral capsid is derived from a virus with a tropism
to a
different genus of organisms than the cell. In another embodiment, the viral
capsid is
derived from a virus with a tropism to a different species of organisms than
the cell.
In a specific embodiment, the viral capsid is selected from a virus of Group
IV.
In one embodiment, the viral capsid is selected form an icosahedral virus. The
icosahedral virus can be selected from a member of any of the
Papillornavif°idae,
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Totivi~idae, Dieistf~ovi~idae, Hepadnaviridae, Togavi~idiae, Polyomavi~idiae,
Nodavi~idae, TectiviYidae, Levivi~idae, MicYOViridae, Sipovi~idae,
Nodaviridae,
Picornoviridae, PaYVOVi~idae, Calcivi~idae, Tet~avif~idae, and Satellite
viruses.
In a particular embodiment, the sequence will be selected from members of
any one of the taxa that are specific for at least one plant host. In one
embodiment the
icosahedral plant virus species will be a plant-infectious virus species that
is or is a
member of any of the Bunyavi~idae, Reovif°idae, Rhabdovi~idae,
Luteoviridae,
Nanovit~idae, Partitivi~idae, Sequiviridae, Tyrnoviridae, .Ou~miavirus,
Tobacco
Necrosis Virus Satellite, Caulimoviridae, Geminiviridae, Comoviridae,
Sobemovirus,
Tornbusviridae, or B~omoviridae taxa. In one embodiment, the icosahedral plant
virus
species is a plant-infectious virus species that is or is a member of any of
the
Luteoviridae, Nanoviridae, Pa~titivi~idae, Sequiviridae, Tymoviridae,
Ou~miavi~us,
Tobacco Necrosis Virus Satellite, Caulinaovi~idae, Getninivir~idae,
ConaoviYidae,
Sobemovirus, Tombusviridae, or B~omoviridae taxa. In specific embodiments, the
icosahedral plant virus species is a plant infectious virus species that is or
is a member
of any of the Caulimoviridae, Geminivi~idae, Comoviy°idae,
SobenaoviYUS,
Tombusvi~idae, or Bromovi~idae. In more particular embodiments, the
icosahedral
plant virus species will be a plant-infectious virus species that is or is a
member of
any of the Comovi~idae, SobemoviYUS, Tombusvi~idae, or B~omovi~idae. In more
particular embodiments, the icosahedral plant virus species will be a plant-
infectious
virus species that is a member of the Cornovi~idae or Bromoviridae family. In
a
particular embodiment the viral capsid is derived from a Cowpea Mosaic Virus
or a
Cowpea Chlorotic Mottle Virus. In another embodiment, the viral capsid is
derived
from a species of the B~omoviridae taxa. In a specific embodiment, the capsid
is
derived from an Ilarwirus or an Alfarnovirzrs. In a more specific embodiment,
the
capsid is derived from a Tobacco streak virus, or an Alfalfa mosaic virus
(AMV)
(including AMV 1 or AMV 2).
YLP
The icosahedral viral capsid of the invention is non-infective in the host
cells
described. In one embodiment, a virus like particle (VLP) or cage structure is
formed
in the host cell during or after expression of the viral capsid. In one
embodiment, the
VLP or cage structure also includes the peptide of interest, and in a
particular
embodiment, the peptide of interest is expressed on the surface of the VLP.
The
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CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
expression system typically does not contain additional viral proteins that
allow
infectivity of the virus. In a typical embodiment, the expression system
includes a
host cell and a vector which codes for one or more viral capsids and an
operably
linked peptide of interest. The vector typically does not include additional
viral
assembly proteins. The invention is derived from the discovery that viral
capsids
form to a greater extent in certain host cells and allow for more efficient
recovery of
recombinant peptide.
In one embodiment, the VLP or cage structure is a multimeric assembly of
capsids, including from three to about 200 capsids. In one embodiment, the VLP
or
cage structure includes at least 30, at least 50, at least 60, at least 90 or
at least 120
capsids. In another embodiment, each VLP or cage structure includes at least
150
capsids, at least 160, at least 170, or at least 180 capsids.
In one embodiment, the VLP is expressed as an icosahedral structure. In
another embodiment, the VLP is expressed in the same geometry as the native
virus
that the capsid sequence is derived of. In a separate embodiment, however, the
VLP
does not have the identical geometry of the native virus. In certain
embodiments, for
example, the structure is produced in a particle formed of multiple capsids
but not
forming a native-type VLP. For example, a cage structure of as few as 3 viral
capsids
can be formed. In separate embodiments, cage structures of about 6, 9, 12, 15,
18, 21,
24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, or 60 capsids can be formed.
In one embodiment, at least one of the capsids includes at least one peptide
of
interest. In one embodiment, the peptide is expressed within at least one
internal loop,
or in at least one external surface loop of the VLP.
More than one loop of the viral capsid can be modified. In one particular
embodiment, at the recombinant peptide is expressed on at least two surface
loops of
the icosahedal virus-like particle. In another embodiment, at least two
different
peptides are inserted into at least two surface loops of the viral capsid,
cage or virus-
like particle. In another embodiment, at least three recombinant peptides are
inserted
into at least three surface loops of the virus-like particle. The recombinant
peptides in
the surface loops can have the same amino acid sequence. In separate
embodiments,
the amino acid sequence of the recombinant peptides in the surface loops
differs.
In certain embodiments, the host cell can be modified to improve assembly of
the VLP. The host cell can, for example, be modified to include chaperone
proteins
that promote the formation of VLPs from expressed viral capsids. In another
19


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embodiment, the host cell is modified to include a repressor protein to more
efficiently regulate the expression of the capsid to promote regulated
formation of the
VLPs.
The nucleic acid sequence encoding the viral capsid or proteins can also be
additionally modified to alter the formation of VLPs (see e.g. Brumfield, et
al. (2004)
J. Gen. Viol. 85: 1049-1053). For example, three general classes of
modification are
most typically generated for modifying VLP expression and assembly. These
modifications are designed to alter the interior, exterior or the interface
between
adj acent subunits in the assembled protein cage. To accomplish this,
mutagenic
primers can be used to: (i) alter the interior surface charge of the viral
nucleic acid
binding region by replacing basic residues (e.g. K, R) in the N terminus with
acidic
glutamic acids (Douglas et al., 2002b); (ii) delete interior residues from the
N
terminus (in CCMV, usually residues 4-37); (iii) insert a cDNA encoding an 11
amino
acid peptide cell-targeting sequence (Graf et al., 1987) into a surface
exposed loop ;
and (iv) modify interactions between viral subunits by altering the metal
binding sites
(in CCMV, residues 81/148 mutant).
Recombifaant Peptides
Size
In one embodiment, the peptides operably linked to a viral capsid sequence
contain at least two amino acids. In another embodiment, the peptides are at
least
three, at least four, at least five, or at least six amino acids in length. In
a separate
embodiment, the peptides are at least seven amino acids long. The peptides can
also
be at least eight, at least nine, at least ten, at least 11, 12, 13, 14, 15,
16, 17, 18, 19, 20,
30, 45, 50, 60, 65, 75, 85, 95, 96, 99 or more amino acids long. Ili one
embodiment,
the peptides encoded are at least 25kD.
In one embodiment, the peptide will contain from 2 to about 300 amino acids,
or about 5 to about 250 amino acids, or about 5 to about 200 amino acids, or
about 5
to about 150 amino acids, or about 5 to about 100 amino acids. In another
embodiment, the peptide contains or about 10 to about 140 amino acids, or
about 10
to about 120 amino acids, or about 10 to about 100 amino acids.


CA 02547511 2006-05-29
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In one embodiment, the peptides or proteins operably linked to a viral capsid
sequence will contain about 500 amino acids. In one embodiment, the peptide
will
contain less than 500 amino acids. In another embodiment, the peptide will
contain up
to about 300 amino acids, or up to about 250, or up to about 200, or up to
about 180,
or up to about 160, or up to about 150, or up to about 140, or up to about
120, or up to
about 110, or up to about 100, or up to about 90, or up to about 80, or up to
about 70,
or up to about 60, or up to about 50, or up to about 40 or up to about 30
amino acids.
In one embodiment, the recombinant peptide fused to the icosahedral capsid is
at least 7, at least 8, at least, 9, at least 10, at least 12, at least 15, at
least 20, at least
25, at least 30, at least 35, at least 40, at least 45, at least 50, at least
55, at least 60, at
least 65, at least 75, at least 85, at least 95, at least 99, or at least 100
amino acids.
In one embodiment of the present invention, the recombinant peptide contains
at least one monomer of a desired target peptide. In an alternative
embodiment, the
recombinant peptide contains more than one monomer of a desired target
peptide. In
certain embodiments, the peptide is composed of at least two, at least 5, at
least 10, at
least 15 or at least 20 separate monomers that are operably linked as a
concatameric
peptide to the capsid. In another embodiment, the individual monomers in the
concatameric peptide are linked by cleavable linker regions. In still another
embodiment, the recombinant peptide is inserted into at least one surface loop
of the
icosahedral virus-like particle. In one embodiment, at least one monomer is
inserted
in a surface loop of the virus-like particle.
Classification
The peptides of interest that are fused to the viral capsids can be a
heterologous protein that is not derived from the virus and, optionally, that
is not
derived from the same species as the cell.
The peptides of interest that are fused to the viral capsids can be functional
peptides; structural peptides; antigenic peptides, toxic peptides,
antimicrobial peptides,
fragments thereof; precursors thereof; combinations of any of the foregoing;
and/or
concatamers of any of the foregoing. In one embodiment of the present
invention, the
recombinant peptide is a therapeutic peptide useful for human and animal
treatments.
Functional peptides include, but are not limited to, e.g.: bio-active peptides
(i.e.
peptides that exert, elicit, or otherwise result in the initiation,
enhancement,
prolongation, attenuation, termination, or prevention of a biological function
or
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activity in or of a biological entity, e.g., an organism, cell, culture,
tissue, organ, or
organelle); catalytic peptides; microstructure- and nanostructure-active
peptides (i.e.
peptides that form part of engineered micro- or nano-structures in which, or
in
conjunction with which, they perform an activity, e.g., motion, energy
transduction);
and stimulant peptides (e.g., peptide flavorings, colorants, odorants,
pheromones,
attractants, deterrents, and repellants).
Bio-active peptides include, but are not limited to, e.g.: immunoactive
peptides
(e.g., antigenic peptides, allergenic peptides, peptide immunoregulators,
peptide
immunomodulators); signaling and signal transduction peptides (e.g., peptide
hormones, cytokines, and neurotransmitters; receptors; agonist and antagonist
peptides; peptide targeting and secretion signal peptides); and bio-inhibitory
peptides
(e.g., toxic, biocidal, or biostatic peptides, such as peptide toxins and
antimicrobial
peptides).
Structural peptides include, but are not limited to, e.g.: peptide aptamers;
folding peptides (e.g., peptides promoting or inducing formation or retention
of a
physical conformation in another molecule); adhesion-promoting peptides (e.g.,
adhesive peptides, cell-adhesion-promoting peptides); interfacial peptides
(e.g.,
peptide surfactants and emulsifiers); microstructure and nanostructure-
architectural
peptides (i.e. structural peptides that form part of engineered micro- or nano-

structures); and pre-activation peptides (e.g., leader peptides of pre-, pro-,
and pre-
pro-proteins and -peptides; inteins).
Catalytic Peptides include, e.g., apo B RNA-editing cytidine deaminase
peptides; catalytic peptides of glutaminyl-tRNA synthetases; catalytic
peptides of
aspartate transcarbamoylases; plant Type 1 ribosome-inactivating peptides;
viral
catalytic peptides such as, e.g., the foot-and-mouth disease virus [FMDV-2A]
catalytic peptide; matrix metalloproteinase peptides; and catalytic metallo-
oligopeptides.
The peptide can also be a peptide epitopes, haptens, or a related peptides
(e.g.,
antigenic viral peptides; virus related peptides, e.g., HIV-related peptides,
hepatitis
related peptides; antibody idiotypic domains; cell surface peptides; antigenic
human,
animal, protist, plant, fungal, bacterial, and/or archaeal peptides;
allergenic peptides
and allergen desensitizing peptides).
The peptide can also be a peptide immunoregulators or immunomodulators
(e.g., interferons, interleukins, peptide immunodepressants and
immunopotentiators);
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an antibody peptides (e.g., single chain antibodies; single chain antibody
fragments
and constructs, e.g., single chain Fv molecules; antibody light chain
molecules,
antibody heavy chain molecules, domain-deleted antibody light or heavy chain
molecules; single chain antibody domains and molecules, e.g., a CHl, CH1-3,
CH3,
CH1-4, CH4, VHCHl, CL, CDR1, or FRl-CDR1-FR2 domain; paratopic peptides;
microantibodies); another binding peptide (e.g., peptide aptamers,
intracellular and
cell surface receptor proteins, receptor fragments; anti-tumor necrosis factor
peptides).
The peptide can also be an enzyme substrate peptide or an enzyme inhibitor
peptide (e.g., caspase substrates and inhibitors, protein kinase substrates
and inhibitors,
fluorescence-resonance-energy transfer-peptide enzyme substrates).
The peptide can also be a cell surface receptor peptide ligand, agonist, and
antagonist (e.g., caeruleins, dynorphins, orexins, pituitary adenylate cyclase
activating
peptides, tumor necrosis factor peptides; synthetic peptide ligands, agonists,
and
antagonists); a peptide hormone (e.g., endocrine, paracrine, and autocrine
hormones,
including, e.g.: amylins, angiotensins, bradykinins, calcitonins,
cardioexcitatory
neuropeptides, casomorphins, cholecystokinins, corticotropins and
corticotropin-
related peptides, differentiation factors, endorphins, endothelins,
enkephalins,
erythropoietins, exendins, follicle-stimulating hormones, galanins, gastrins,
glucagons
and glucagon-like peptides, gonadotropins, growth hormones and growth factors,
insulins, kallidins, kinins, leptins, lipotropic hormones, luteinizing
hormones,
melanocyte stimulating hormones, melatonins, natriuretic peptides,
neurokinins,
neuromedins, nociceptins, osteocalcins, oxytocins (i.e. ocytocins),
parathyroid
hormones, pleiotrophins, prolactins, relaxins, secretins, serotonins, sleep-
inducing
peptides, somatomedins, thymopoietins, thyroid stimulating hormones,
thyrotropins,
urotensins, vasoactive intestinal peptides, vasopressins); a peptide cytokine,
chemokine, virokine, and viroceptor hormone releasing and release-inhibiting
peptide
(e.g., corticotropin-releasing hormones, cortistatins, follicle-stimulating-
hormone-
releasing factors, gastric inhibitory peptides, gastrin releasing peptides,
gonadotropin-
releasing hormones, growth hormone releasing hormones, luteinizing hormone-
releasing hormones, melanotropin-releasing hormones, melanotropin-release
inhibiting factors; nocistatins, pancreastatins, prolactinreleasing peptides,
prolactin
release-inhibiting factors; somatostatins; thyrotropin releasing hormones); a
peptide
neurotransmitter or channel blocker (e.g., bombesins, neuropeptide Y,
neurotensins,
substance P) a peptide toxin, toxin precursor peptide, or toxin peptide
portion. In
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certain embodiments, a peptide toxin contains no D-amino acids. Toxin
precursor
peptides can be those that contain no D-amino acids and/or that have not been
converted by posttranslational modification into a native toxin structure,
such as, e.g.,
by action of a D configuration inducing agent (e.g., a peptide isomerase(s) or
epimeras(e) or racemase(s) or transaminase(s)) that is capable of introducing
a D-
configuration in an amino acid(s), and/or by action of a cyclizing agent
(e.g., a peptide
thioesterase, or a peptide ligase such as a trans-splicing protein or intein)
that is
capable of form a cyclic peptide structure.
Toxin peptide portions can be the linear or pre-cyclized oligo- and poly-
peptide portions of peptide-containing toxins. Examples of peptide toxins
include, e.g.,
agatoxins, amatoxins, charybdotoxins, chlorotoxins, conotoxins, dendrotoxins,
insectotoxins, margatoxins, mast cell degranulating peptides, saporins,
sarafotoxins;
and bacterial exotoxins such as, e.g., anthrax toxins, botulism toxins,
diphtheria toxins,
and tetanus toxins.
The peptide can also be a metabolism- and digestion-related peptide (e.g.,
cholecystokinin-pancreozymin peptides, peptide yy, pancreatic peptides,
motilins); a
cell adhesion modulating or mediating peptide, extracellular matrix peptide
(e.g.,
adhesins, selectins, laminins); a neuroprotectant or myelination-promoting
peptide; an
aggregation inhibitory peptide (e.g., cell or platelet aggregation inhibitor
peptides,
amyloid formation or deposition inhibitor peptides); a joining peptide (e.g.,
cardiovascular joining neuropeptides, iga joining peptides); or a
miscellaneous
peptide (e.g., agouti-related peptides, amyloid peptides, bone-related
peptides, cell-
permeable peptides, conantokins, contryphans, contulakins, myelin basic
protein, and
others).
In certain embodiments, the peptide of interest is exogenous to the selected
viral capsid. Peptides may be either native or synthetic in sequence (and
their coding
sequences rnay be either native or synthetic nucleotide sequences). Thus,
e.g., native,
modified native, and entirely artificial sequences of amino acids are
encompassed.
The sequences of the nucleic acid molecules encoding these amino acid
sequences
likewise may be native, modified native, or entirely artificial nucleic acid
sequences,
and may be the result of, e.g., one or more rational or random mutation and/or
recombination and/or synthesis and/or selection process employed (i. e.
applied by
human agency) to obtain the nucleic acid molecules.
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The coding sequence can be a native coding sequence for the target peptide, if
available, but will more typically be a coding sequence that has been
selected,
improved, or optimized for use in the selected expression host cell: for
example, by
synthesizing the gene to reflect the codon use preference of a host species.
In one
embodiment of the invention, the host species is a P. fZuorescens, and the
codon
preference of P. fluoreseeras is taken into account when designing both the
signal
sequence and the peptide sequence
Aratigeraic Peptides (Peptide Epitopes)
In one embodiment, an antigenic peptide is produced through expression with
a viral capsid. The antigenic peptide can be selected from those that are
antigenic
peptides of human or animal pathogenic agents, including infectious agents,
parasites,
cancer cells, and other pathogenic agents. Such pathogenic agents also include
the
virulence factors and pathogenesis factors, e.g., exotoxins, endotoxins, et
al., of those
agents. The pathogenic agents may exhibit any level of virulence, i.e. they
may be,
e.g., virulent, avirulent, pseudo-virulent, semi-virulent, and so forth. In
one
embodiment, the antigenic peptide will contain an epitopic amino acid sequence
from
the pathogenic agent(s). In one embodiment, the epitopic amino acid sequence
will
include that of at least a portion of a surface peptide of at least one such
agent. . In
one embodiment, the capsid-recombinant peptide virus like particles can be
used as a
vaccine in a human or animal application.
More than one antigenic peptide may be selected, in which case the resulting
virus-like particles can present multiple different antigenic peptides. In a
particularly
embodiment of a multiple antigenic peptide format, the various antigenic
peptides will
all be selected from a plurality of epitopes from the same pathogenic agent.
In a
particular embodiment of a multi-antigenic-peptide format, the various
antigenic
peptides selected will all be selected from a plurality of closely related
pathogenic
agents, for example, different strains, subspecies, biovars, pathovars,
serovars, or
genovars of the same species or different species of the same genus.
In one embodiment, the pathogenic agents) will belong to at least one of the
following groups: Bacteria and Mycoplasma agents including, but not limited
to,
pathogenic: Bacillus spp., e.g., Bacillus anthr~acis; Bartonella spp., e.g.,
B. quintarra;
Br°ucella spp.; BuYklrolderia spp., e.g., B. pseudornallei;
Campylobacter spp.;
Clostr~idiurn spp., e.g., C. tetani, C. botulinuna; Coxiella spp., e.g., C.
burnetii;


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Edwardsiella spp., e.g., E. tarda; Enterobacter spp., e.g., E. cloacae;
Enterococcus
spp., e.g., E. faecalis, E. faecium; Escherichia spp., e.g., E. coli;
Francisella spp., e.g.,
F. tularensis; Haemoplailus spp., e.g., H. infZuenzae; Klebsiella spp., e.g.,
K.
pneumoniae; Legionella spp.; Listeria spp., e.g., L. monocytogenes;
Meningococci
and Gonococci, e.g., Neisseria spp.; Moraxella spp.; Mycobacteriurn spp.,
e.g., M.
leprae, M. tuberculosis; Pneumococci, e.g., Diplococcus pneuntoniae;
Pseudomonas
spp., e.g., P. aeruginosa; Rickettsia spp., e.g., R. prowazekii, R.
rickettsii, R. typlai;
Salmotaella spp., e.g., S. typhi; Staphylococcus spp., e.g., S. aureus;
Streptococcus
spp., including Group A Streptococci and hemolytic Streptococci, e.g., S.
pneumoniae,
S. pyogenes; Streptomyces spp.; Slaigella spp.; Tlibrio spp., e.g., Tl
cholerae; and
Yersinia spp., e.g., Y. pestis, Y. enterocolitica. Fungus and Yeast agents
including, but
not limited to, pathogenic: Alternaria spp.; Aspergillus spp.; Blastonayces
spp., e.g., B.
dermatiditis; Candida spp., e.g., C. albicans; Cladosporium spp.; Coccidiodes
spp.,
e.g., C. immitis; Cryptococcus spp., e.g., C. neoformans; Histoplasma spp.,
e.g., H.
capsulatum; and Sporotlarix spp., e.g., S schenckzi.
In one embodiment, the pathogenic agents) will be from a protist agent
including, but not limited to, pathogenic: Amoebae, including Acanthamoeba
spp.,
Amoeba spp., Naegleria spp., Entamoeba spp., e.g., E. laistolytica;
Cfyptosporidiurn
spp., e.g., C. parvum; Cyclospora spp.; Encephalitozoon spp., e.g., E.
intestinalis;
Enterocytozoon spp.; Giardia spp., e.g., G. laznblia; Isospora spp.;
Microsporidiunz
spp.; Plasmodium spp., e.g., P. falciparuna, P. malariae, P. ovule, P. vivax;
Toxoplasma spp., e.g., T. gondii; and Tzypanosoma spp., e.g., T. brucei.
In one embodiment, the pathogenic agents) will be from a parasitic agent
(e.g.,
helininthic parasites) including, but not limited to, pathogenic: Ascaris
spp., e.g., A.
lumbricoides; Dracunculus spp., e.g., D. naedinertsis; Onchocerca spp., e.g.,
O.
volvulus; Sclaistosoma spp.; Trichinella spp., e.g., T. spiralis; and
Trichuris spp., e.g.,
T. trichiura.
In another embodiment, the pathogenic agents) will be from a viral agent
including, but not limited to, pathogenic: Adenoviruses; Arenaviruses, e.g.,
Lassa
Fever viruses; Astroviruses; Bunyaviruses, e.g., Hantaviruses, Rift Valley
Fever
viruses; Coronaviruses, Deltaviruses; Cytomegaloviruses, Epstein-Barr viruses,
Herpes viruses, Varicella viruses; Filoviruses, e.g., Ebola viruses, Marburg
viruses;
Flaviruses, e.g., Dengue viruses, West Nile Fever viruses, Yellow Fever
viruses;
Hepatitis viruses; Influenzaviruses; Lentiviruses, T-Cell Lymphotropic
viruses, other
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leukemia viruses; Norwalk viruses; Papillomaviruses, other tumor viruses;
Paramyxoviruses, e.g., Measles viruses, Mumps viruses, Parainfluenzaviruses,
Pneumoviruses, Sendai viruses; Parvoviruses; Picornaviruses, e.g.,
Cardioviruses,
Coxsackie viruses, Echoviruses, Poliomyelitis viruses, Rhinoviruses, Other
Enteroviruses; Poxviruses, e.g., Variola viruses, Vaccinia viruses,
Parapoxviruses;
Reoviruses, e.g., Coltiviruses, Orbiviruses, Rotaviruses; Rhabdoviruses, e.g.,
Lyssaviruses, Vesicular Stomatitis viruses; and Togaviruses, e.g., Rubella
viruses,
Sindbis viruses, Western Encephalitis viruses.
In one particular embodiment, the antigenic peptide is selected from the group
consisting of a Canine parvovirus peptide, Bacillus anthracis protective
antigen (PA)
antigenic peptide, and an Eastern Equine Encephalitis virus antigenic peptide.
In a
particular embodiment, the antigenic peptide is the canine parvovirus-derived
peptide
with the amino acid sequence of SEQ. m. NO: 7. In another particular
embodiment,
the antigenic peptide is the Bacillus anthracis protective antigen (PA)
antigenic
peptide with any one of the amino acid sequence of SEQ. ~. NOs: 9, 11, 13 or
15. In
still another particular embodiment, the antigenic peptide is an Eastern
equine
Encephalitis virus antigenic peptide with the amino acid sequence of one of
SEQ. m.
NOs:25 or 27.
Host-Cell Toxic Peptide
In another particular embodiment, the recombinant peptide is a peptide that is
toxic to the host cell when in free monomeric form. In a more particular
embodiment,
the toxic peptide is an antimicrobial peptide.
In certain embodiments, the peptide of interest expressed in conjunction with
a
viral capsid will be a host cell toxic peptide. In certain embodiments, this
protein will
be an antimicrobial peptide. A host cell toxic peptide indicates a bio-
inhibitory
peptide that is biostatic, biocidal, or toxic to the host cell in which it is
expressed, or
to other cells in the cell culture or organism of which the host cell is a
member, or to
cells of the organism or species providing the host cells. In one embodiment,
the host-
cell-toxic peptide will be a bioinhibitory peptide that is biostatic,
biocidal, or toxic to
the host cell in which it is expressed. Some examples of host-cell-toxic
peptides
include, but are not limited to: peptide toxins, anti-microbial peptides, and
other
antibiotic peptides.
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Anti-Microbial Peptides include, e.g., anti-bacterial peptides such as, e.g.,
magainins, betadefensins, some alpha-defensins; cathelicidins; histatins; anti-
fungal
peptides; antiprotozoal peptides; synthetic AMPS; peptide antibiotics or the
linear or
pre-cyclized oligo- or poly-peptide portions thereof; other antibiotic
peptides (e.g.,
anthelinintic peptides, hemolytic peptides, tumoricidal peptides); and anti-
viral
peptides (e.g., some alpha-defensins; virucidal peptides; peptides that
inhibit viral
infection). In one particular embodiment, the antimicrobial peptide is the
D2A21
peptide with the amino acid sequence of SEQ ID N0:20. In another embodiment,
the
antimicrobial peptide is antimicrobial peptide PBF20 with the amino acid
sequence
corresponding substantially to SEQ ID N0:24.
Cells fog use in Expressiyzg the TILP
The cell used as a host for the expression of the viral capsid or viral capsid
fusion peptide (also referred to as "host cell") of the invention will be one
in which
the viral capsid does not allow replication or infection of the cell. In one
embodiment,
the viral capsid will be derived from a virus that does not infect the species
of cell that
the host cell is derived from. For example, in one embodiment, the viral
capsid is
derived from an icosahedral plant virus and is expressed in a host cell of a
bacterial
species. In another embodiment, the viral species infects mammals and the
expression system includes a bacterial host cell.
In one embodiment, the host cell can be a prokaryote such as a bacterial cell
including, but not limited to a Pseudo~raonas species. Typical bacterial cells
are
described, for example, in "Biological Diversity: Bacteria and Archaeans", a
chapter
of the On-Line Biology Book, provided by Dr MJ Farabee of the Estrella
Mountain
Community College, Arizona, USA at URL: http://www.emc.maricopa.edu/facultyl
farabeelBIOBK/BioBookDiversity _ 2.html. In certain embodiments, the host cell
can be a Pseudomonad cell, and can typically be a P. fluof~escefzs cell.
In one embodiment, the host cell can be a member of any species of eubacteria.
The host can be a member any one of the taxa: Acidobacteria, Actinobacteira,
Aquificae, Bacteroidetes, Chlorobi, Chlamydiae, Choroflexi, Chrysiogenetes,
Cyanobacteria, Defernbacteres, Deinococcus, Dictyoglomi, Fibrobacteres,
Firmicutes,
Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospirae, Planctomycetes,
Proteobacteria, Spirochaetes, Thermodesulfobacteria, Thermomicrobia,
Thermotogae,
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Thermus (Thermales), or Verrucomicrobia. In an embodiment of a eubacterial
host
cell, the cell can be a member of any species of eubacteria, excluding
Cyanobacteria.
The bacterial host can also be a member of any species of Proteobacteria. A
proteobacterial host cell can be a member of any one of the taxa
Alphaproteobacteria,
Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, or
Epsilonproteobacteria. In addition, the host can be a member of any one of the
taxa
Alphaproteobacteria, Betaproteobacteria, or Gamrnaproteobacteria, and a member
of
any species of Gammaproteobacteria.
In one embodiment of a Gamma Proteobacterial host, the host will be member
of any one of the taxa Aeromonadales, Altet°omofaadales,
Enterobacteriales,
Pseudomonadales, or Xantl2onaonadales; or a member of any species of the
EnteYObacteniales or Pseudonaonadales. In one embodiment, the host cell can be
of
the order Enterobacteriales, the host cell will be a member of the family
Entef°obacteriaceae, or a member of any one of the genera Enwinia ,
Esclzenichia, or
Sernatia; or a member of the genus Esclzenichia. In one embodiment of a host
cell of
the order Pseudomonadales, the host cell will be a member of the family
Pseudomonadaceae, even of the genus Pseudomonas . Gamma Proteobacterial hosts
include members of the species Esche~iclaia coli and members of the species
Pseudonaonas fluonescens.
Other Pseudomonas organisms may also be used. Pseudomonads and closely
related species include Gram(-) Proteobacteria Subgroup l, which include the
group
of Proteobacteria belonging to the families and/or genera described as "Gram-
Negative Aerobic Rods and Cocci" by R.E. Buchanan and N.E. Gibbons (eds.),
Bergey's Manual of Determinative Bacteriology, pp. 217-289 (8th ed., 1974)
(The
Williams & Will~ins Co., Baltimore, MD, USA) (hereinafter "Bergey (1974)").
Table
1 presents these families and genera of organisms.
TABLE 1. FAMILIES AND GENERA LISTED IN THE PART, "GRAM-NEGATIVE
AEROBIC RODS AND COCCI" (1N BERGEY (1974))
amily I. Pseudomonadaceae ~Gluconobacter
29


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WO 2005/067478 PCT/US2004/040117
amily II. Azotobacteraceae Azomonas


zotobacte~


eijerirackia


e~xia


amily III. Rhizobiaceae g~obacte~ium


hizobiuna


amity 1V. Methylomonadaceae ethylococcus


etlaylonzonas


amity V. Halobacteriaceae alobacterium


alococcus


Other Genera cetobacter


lcaligenes


ordetella


rucella


~ancisella


Thernaus


"Gram(-) Proteobacteria Subgroup 1" also includes Proteobacteria that would
be classified in this heading according to the criteria used in the
classification. The
heading also includes groups that were previously classified in this section
but are no
longer, such as the genera Acidovo~ax, Brevundimonas, Burkholdef°ia,
Hydrogenophaga, Oceanimotaas, Ralstonia, and Stenot~oplaonao~Zas, the genus
Sphingomonas (and the genus Blastonaonas, derived therefrom), which was
created
by regrouping organisms belonging to (and previously called species of) the
genus
Xanthomonas, the genus Acidonaonas, which was created by regrouping organisms
belonging to the genus Acetobacter as defined in Bergey (1974). In addition
hosts
can include cells from the genus Pseudomonas , Pseudomonas enalia (ATCC
14393),
Pseudotnonas nigrifaciens (ATCC 19375), and Pseudonaonas putrefaciens (ATCC
8071), which have been reclassified respectively as Alter~omonas haloplanktis,
Alter~onaonas nig~ifaciens, and Alte~onaonas putrefaciens. Similarly, e.g.,
Pseudornonas acidovo~ans (ATCC 15668) and Pseudonaonas testosterofai (ATCC
11996) have since been reclassified as Comamonas acidovo~ans and Cornamonas
testosterone, respectively; and Pseudomonas nig~ifaciens (ATCC 19375) and
Pseudornonas piscicida (ATCC 15057) have been reclassified respectively as
Pseudoalteromonas nigr~ifaciens and Pseudoalteromonas piscicida. "Gram(-)
Proteobacteria Subgroup 1" also includes Proteobacteria classified as
belonging to
any of the families: Pseudomonadaceae, Azotobacteraceae (now often called by
the
synonym, the "Azotobacter group" of Pseudomonadaceae), Rhizobiaceae, and
Methylomonadaceae (now often called by the synonym, " Methylococcaceae").


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
Consequently, in addition to those genera otherwise described herein, further
Proteobacterial genera falling within "Gram(-) Proteobacteria Subgroup 1"
include:
1) Azotobacter group bacteria of the genus Azo>"hizophilus; 2)
Pseudomonadaceae
family bacteria of the genera Cellvibz~io, Oligella, and Teredinibacter; 3)
Rhizobiaceae family bacteria of the genera Chelatobacter, Ensifer,
Liberibacter (also
called "Candidatus Liberibacte>~"), and Sinorhizobiunz; and 4)
Methylococcaceae
family bacteria of the genera Methylobacter, Metlzylocaldum, Methylomicrobium,
Methylosarcizza, and Methylosphaera.
In another embodiment, the host cell is selected from "Gram(-) Proteobacteria
Subgroup 2." "Gram(-) Proteobacteria Subgroup 2" is defined as the group of
Proteobacteria of the following genera (with the total numbers of catalog-
listed,
publicly-available, deposited strains thereof indicated in parenthesis, all
deposited at
ATCC, except as otherwise indicated): Acidomonas (2); Acetobacte>" (93);
Gluconobacter (37); Brevundimozzas (23); Beije>~inckia (13); Dez~xia (2);
B~ucella (4);
Agrobactel"ium (79); Chelatobactez~ (2); Ensife>" (3); Rhizobiuzn (144);
Sizzorlzizobium
(24); Blastotnozzas (1); Splzingomonas (27); Alcaligenes (88); Bordetella
(43);
Burklzolderia (73); Ralstonia (33); Acidovoz°ax (20); Hydrogenophaga
(9); Zoogloea
(9); Methylobaeten (2); Methylocaldum (1 at NCIMB); Metlzylococcus (2);
Methylomic~obium (2); Methylomonas (9); Methylosaz°cina (1);
Metlzylosphaera;
Azomonas (9); Azorhizophilus (5); Azotobacten (64); Cellvibf~io (3); Oligella
(5);
Pseudomozzas (1139); Frazzcisella (4); Xanthomonas (229); Stenotz"ophoznozzas
(50);
and Oceanimonas (4).
Exemplary host cell species of "Gram(-) Proteobacteria Subgroup 2" include,
but are not limited to the following bacteria (with the ATCC or other deposit
numbers
of exemplary strains) thereof shown in parenthesis): Acidomonas methanolica
(ATCC 43581); Acetobacter aceti (ATCC 15973); Gluconobacter oxydazzs (ATCC
19357); Brevundiznonas dizninuta (ATCC 11568); Beijerinckia indica (ATCC 9039
and ATCC 19361); Derxia guznmosa (ATCC 15994); Brucella melitensis (ATCC
23456), Brucella abortus (ATCC 23448); Agrobactez~iunz tuznefaciens (ATCC
23308),
Agrobactez°iunz ~adiobacter (ATCC 19358), Agrobacterium
rlzizogenes (ATCC
11325); ClzelatobacteY heizztzii (ATCC 29600); Ezzsifez° adhae>~ens
(ATCC 33212);
Rhizobiunz leguminosa~um (ATCC 10004); SinoYhizobiunz f-edii (ATCC 35423);
Blastomonas natatof~ia (ATCC 35951); Sphingonzozzas pauciznobilis (ATCC
29837);
Alcaligenes faecalis (ATCC 8750); Bo~detella pertussis (ATCC 9797);
Burkholdenia
31


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cepacia (ATCC 25416); Ralstonia pickettii (ATCC 27511); Acidovorax facilis
(ATCC 11228); Hydrogerzophaga flava (ATCC 33667); Zoogloea ramigera (ATCC
19544); Methylobacter luteus (ATCC 49878); Methylocaldum gracile (NCIMB
11912); Methylococcus capsulatus (ATCC 19069); Methylomic~obium agile (ATCC
35068); Methylomonas methanica (ATCC 35067); Metlzylosarcina fibr~ata (ATCC
700909); Methylosphaera hansonii (ACAM 549); Azonzonas agilis (ATCC 7494);
AzoYhizophilus paspali (ATCC 23833); Azotobacter chr~oococcum (ATCC 9043);
Cellvibrio rnixtus (UQM 2601); Oligella uretltralis (ATCC 17960);
Pseudornortas
aerwginosa (ATCC 10145), Pseudomonas fluor~escens (ATCC 35858); Fr~artcisella
tularertsis (ATCC 6223); Stenotrophomonas maltophilia (ATCC 13637);
Xanthomonas campestr°is (ATCC 33913); and Oceanirnorzas doudoro~i
(ATCC
27123).
In another embodiment, the host cell is selected from "Gram(-) Proteobacteria
Subgroup 3." "Gram(-) Proteobacteria Subgroup 3" is defined as the group of
Proteobacteria of the following genera: Brevundirnonas; Agrobacter~ium;
Rhizobium;
Sinor°hizobiunz; Blastornonas; Sphirtgonzonas; Alcaligenes;
Buz~kholder~ia; Ralstonia ;
Acidovorax; Hydrogenophaga; Methylobacter~; Methylocaldum; Methylococcus;
Metlzylomicrobium; Methylomorzas; Methylosar°cina ; Methylospltaer~a;
Azornonas;
Azorhizoplzilus; Azotobacter-; Cellvibr~io; Oligella; Pseudomonas ;
Ter°edinibacte>";
Fr~ancisella; Stenotrophotnonas; ~Yanthomonas; and Oceanimonas.
In another embodiment, the host cell is selected from "Gram(-) Proteobacteria
Subgroup 4." "Gram(-) Proteobacteria Subgroup 4" is defined as the group of
Proteobacteria of the following genera: Brevundirnonas; Blastomonas;
Spltingontonas ; Burkholder°ia; Ralstorzia; Acidovorax;
Hyd>~ogenoplzaga;
Methylobacter~; Metlzylocaldum; Methylococcus; Methylornicrobium;
Methylomonas;
Methylosa>~cirza; Methylosphaer~a; Azornonas; Azorhizophilus; Azotobacter~;
Cellvibrio; Oligella; Pseudornonas ; TeYedinibacter~; Frartcisella ;
Stenotrophomortas; Xanthornonas; and Oceanirnonas.
In an embodiment, the host cell is selected from "Gram(-) Proteobacteria
Subgroup 5." "Gram(-) Proteobacteria Subgroup 5" is defined as the group of
Proteobacteria of the following genera: Metlzylobacter; Methylocaldum;
Methylococcus; Methylomicr~obium; Metlzylomonas; Methylosarcina;
Methylosphaera; Azomorzas; Azor~hizophilus; Azotobacter~; Cellvibrio;
Oligella;
32


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Pseudoznonas ; Te~edinibacter; Francisella; Stenotrophomotzas; Xantlzornonas;
and
Oceanimonas.
The host cell can be selected from "Gram(-) Proteobacteria Subgroup 6."
"Gram(-) Proteobacteria Subgroup 6" is defined as the group of Proteobacteria
of the
following genera: Brevundimonas; Blastomonas; Sphingomonas; Bu~kholderia;
Ralstonia; Acidovorax; Hyd~ogeraophaga; Azornonas; Azorhizophilus;
Azotobacter;
Cellvib~io; Oligella; Pseudomonas ; Teredinibactet~; Stenotrophomonas;
Xanthomonas; and Oceanimonas.
The host cell can be selected from "Gram(-) Proteobacteria Subgroup 7."
"Gram(-) Proteobacteria Subgroup 7" is defined as the group of Proteobacteria
of the
following genera: Azomonas; Azo~laizophilus; Azotobacte~; Cellvibrio;
Oligella;
Pseudomonas ; Teredinibactef; Sterzotrophornonas; Xanthornonas; and
Oceanimonas.
The host cell can be selected from "Gram(-) Proteobacteria Subgroup 8."
"Gram(-) Proteobacteria Subgroup 8" is defined as the group of Proteobacteria
of the
following genera: Brevuzzdimonas; Blastomonas; Sphingomonas; Bu~kholderia;
Ralstonia; Acidovoz°ax; Hydf~ogenophaga; Pseudomonas ;
Ste~zotf°ophomonas;
Xanthomonas; and Oceanimonas.
The host cell can be selected from "Gram(-) Proteobacteria Subgroup 9."
"Gram(-) Proteobacteria Subgroup 9" is defined as the group of Proteobacteria
of the
following genera: Bf-evundiynonas; Burkholde~ia; Ralstonia; Acidovorax;
Hydz~ogenophaga; Pseudomonas ; Stenot~ophomonas; and Oceanimonas.
The host cell can be selected from "Gram(-) Proteobacteria Subgroup 10."
"Gram(-) Proteobacteria Subgroup 10" is defined as the group of Proteobacteria
of the
following genera: Burkholderia; Ralstonia; Pseudomonas ; Stezzotrophonzonas;
and
Xanthomonas.
The host cell can be selected from "Gram(-) Proteobacteria Subgroup 11."
"Gram(-) Proteobacteria Subgroup 11" is defined as the group of Proteobacteria
of the
genera: Pseudomonas ; Stenotr~ophomoraas; and Xanthomofzas. The host cell can
be
selected from "Gram(-) Proteobacteria Subgroup 12." "Gram(-) Proteobacteria
Subgroup 12" is defined as the group of Proteobacteria of the following
genera:
Buf°ldzolderia; Ralstonia; Pseudomozzas . The host cell can be selected
from "Gram(-)
Proteobacteria Subgroup 13." "Gram(-) Proteobacteria Subgroup 13" is defined
as the
group of Proteobacteria of the following genera: Burlzholder~ia; Ralstonia;
Pseudonzonas ; and Xanthonaonas. The host cell can be selected from "Gram(-)
33


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
Proteobacteria Subgroup 14." "Gram(-) Proteobacteria Subgroup 14" is defined
as the
group of Proteobacteria of the following genera: Pseudomonas and Xantlzomonas.
The host cell can be selected from "Gram(-) Proteobacteria Subgroup 15."
"Gram(-)
Proteobacteria Subgroup 15" is defined as the group of Proteobacteria of the
genus
Pseudomonas .
The host cell can be selected from "Gram(-) Proteobacteria Subgroup 16."
"Gram(-) Proteobacteria Subgroup 16" is defined as the group of Proteobacteria
of the
following Pseudoznonas species (with the ATCC or other deposit numbers of
exemplary strains) shown in parenthesis): Pseudomonas abietaniphila (ATCC
700689); Pseudomonas aeruginosa (ATCC 10145); Pseudozzzonas alcaligenes
(ATCC 14909); Pseudomonas anguilliseptica (ATCC 33660); Pseudomonas
citronellolis (ATCC 13674); Pseudomotzas flavescens (ATCC 51555); Pseudomonas
mendocina (ATCC 25411); Pseudomonas nitroreducens (ATCC 33634);
Pseudoznonas oleovorans (ATCC 8062); Pseudonzonas pseudoalcaligenes (ATCC
17440); Pseudomonas resinovorans (ATCC 14235); Pseudomotzas straminea (ATCC
33636); Pseudotnonas agarici (ATCC 25941); Pseudornonas alcaliphila;
Pseudonzonas alginovora; Pseudonzonas andersonii; Pseudomonas asplenii (ATCC
23835); Pseudonzonas azelaica (ATCC 27162); Pseudomonas beijerinckii (ATCC
19372); Pseudomorzas borealis; Pseudomonas boreopolis (ATCC 33662);
Pseudomonas brassicacearum; Pseudomonas butanovora (ATCC 43655);
Pseudomonas cellulose (ATCC 55703); Pseudornonas aurantiaca (ATCC 33663);
Pseudomozzas clzlororapltis (ATCC 9446, ATCC 13985, ATCC 17418, ATCC
17461); Pseudomonas fragi (ATCC 4973); Pseudomonas lundezzsis (ATCC 49968);
Pseudomonas taetrolens (ATCC 4683); Pseudomonas cissicola (ATCC 33616);
Pseudomonas coronafaciens; Pseudomonas diterpenipltila; Pseudomonas elongate
(ATCC 10144); Pseudomonas flectezzs (ATCC 12775); Pseudornofzas azotofornzans;
Pseudomozzas brenneri; Pseudomonas cedrella; Pseudomozzas corrugate (ATCC
29736); Pseudomonas extrenzorientalis; Pseudornonas fluof°escens (ATCC
35858);
Pseudomozzas gessardii; Pseudonzonas libanensis; Pseudornonas nzandelii (ATCC
700871); Pseudorzzotzas marginalia (ATCC 10844); Pseudomonas znigulae;
Pseudoznonas znucidolens (ATCC 4685); Pseudomonas or ientalis; Pseudomonas
rhodesiae; Pseudomonas synxayttlza (ATCC 9890); Pseudomonas tolaasii (ATCC
33618); Pseudomonas vef°otzii (ATCC 700474); Pseudo»zonas
frederilzsbergensis;
Pseudomonas geniculata (ATCC 19374); Pseudomonas gingeri; Pseudonzonas
34


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
gr~amirzis; Pseudomonas grinaontii; Pseudomonas lzalodenitr~ificans;
Pseudomonas
halophila; Pseudomonas hibiscicola (ATCC 19867); Pseudomonas huttiensis (ATCC
14670); Pseudomonas hydrogenovora; Pseudornoraas jesseraii (ATCC 700870);
Pseudomonas kiloraensis; Pseudomonas lanceolata (ATCC 14669); Pseudomonas
lirZi; Pseudomonas naarginata (ATCC 25417); Pseudomonas rnephitica (ATCC
33665); Pseudomonas denitr~ificans (ATCC 19244); Pseudomonas peYtucirZOgena
(ATCC 190); Pseudornonas pictorum (ATCC 23328); Pseudomonas psychrophila;
Pseudornonas fulva (ATCC 31418); Pseudomonas monteilii (ATCC 700476);
Pseudomonas mosselii; PseudomorZas oryzilZabitans (ATCC 43272); Pseudornonas
plecoglossicida (ATCC 700383); Pseudomonas putida (ATCC 12633); Pseudonaonas
reactans; Pseudomonas spinosa (ATCC 14606); Pseudomonas balearica;
Pseudomonas luteola (ATCC 43273); Pseudornonas stutzer~i (ATCC 17588);
Pseudornonas anaygdali (ATCC 33614); Pseudomonas avellaraae (ATCC 700331);
Pseudomonas caricapapayae (ATCC 33615); Pseudomonas cichorii (ATCC 10857);
PseudomorZas ficuserectae (ATCC 35104); Pseudomoraas fuscovaginae;
Pseudomonas meliae (ATCC 33050); Pseudornonas syringae (ATCC 19310);
Pseudornonas viridiflava (ATCC 13223); Pseudomonas
ther°mocarboxydovorans
(ATCC 35961); Pseudornonas ther~rnotoler°aras; Pseudornonas
thivervalerZSis;
Pseudornonas vancouver°ensis (ATCC 700688); Pseudomonas
wisconsinensis; and
Pseudornonas xianaenensis.
The host cell can be selected from "Gram(-) Proteobacteria Subgroup 17."
"Gram(-) Proteobacteria Subgroup 17" is defined as the group of Proteobacteria
known in the art as the "fluorescent Pseudomonads" including those belonging,
e.g.,
to the following Pseudornonas species: Pseudonaonas azotofo~rnarzs;
Pseudomonas
br~enneYi; Pseudornonas ced>"ella; Pseudonaonas corr°ugata; Pseudomonas
extt~ernor~ierZtalis; Pseudomonas fluor~escens; Pseudomonas gessardii;
Pseudomoraas
libanensis; Pseudonaonas mandelii; Pseudonaoraas marginalis; Pseudonaonas
rnigulae; Pseudonaoraas rnucidolens; Pseudonaonas or ientalis; PseudomorZas
rlaodesiae; Pseudornonas syrZxantha; Pseudonaonas tolaasii; and Pseudornonas
ver~orzii.
In this embodiment, the host cell can be selected from "Gram(-) Proteobacteria
Subgroup 18." "Gram(-) Proteobacteria Subgroup 18" is defined as the group of
all
subspecies, varieties, strains, and other sub-special units of the species
Pseudonaonas
fluorescens, including those belonging, e.g., to the following (with the ATCC
or


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
other deposit numbers of exemplary strains) shown in parenthesis):
Pseudonzonas
fluonescens biotype A, also called biovar 1 or biovar I (ATCC 13525);
Pseudonzonas
fluo~escens biotype B, also called biovar 2 or biovar II (ATCC 17816);
Pseudomonas
fZuorescens biotype C, also called biovar 3 or biovar III (ATCC 17400);
Pseudomofzas
fluof°escens biotype F, also called biovar 4 or biovar IV (ATCC 12983);
Pseudomonas
fluo~escens biotype G, also called biovar 5 or biovar V (ATCC 17518);
Pseudomonas
fluorescens biovar VI; Pseudomonas fluo~escens Pf0-1; Pseudoznonas fZuorescens
Pf
5 (ATCC BAA-477); Pseudomonas fZuof~escens SBW25; and Pseudomonas
fluof~escens subsp. cellulose (NCI1VIB 10462).
The host cell can be selected from "Gram(-) Proteobacteria Subgroup 19."
"Gram(-) Proteobacteria Subgroup 19" is defined as the group of all strains of
PseudomorZas fluo~escens biotype A. A particular strain of this biotype is P.
fZuorescens strain MB101 (see U.S. Patent No. 5,169,760 to Wilcox), and
derivatives
thereof. An example of a derivative thereof is P. fluo~escens strain MB214,
constructed by inserting into the MB101 chromosomal asd (aspartate
dehydrogenase
gene) locus, a native E. coli PlacI-lacI-lacZYA construct (i.e. in which PlacZ
was
deleted).
Additional P. fluof°escens strains that can be used in the present
invention
include Pseudomonas fluorescens Migula and Pseudonzonas fluorescens
Loitokitok,
having the following ATCC designations: [NCIB 8286]; NRRL B-1244; NCIB 8865
strain C01; NCIB 8866 strain C02; 1291 [ATCC 17458; IFO 15837; NCIB 8917;
LA; NRRL B-1864; pyrrolidine; PW2 [ICMP 3966; NCPPB 967; NRRL B-899];
13475; NCTC 10038; NRRL B-1603 [6; IFO 15840]; 52-1C; CCEB 488-A [BU
140]; CCEB 553 [IEM 15/47]; M 1008 [AHH-27]; IAM 1055 [AHH-23]; 1 [IFO
15842]; 12 [ATCC 25323; NIH 11; den Dooren de Jong 216]; 18 [IFO 15833; WRRL
P-7]; 93 [TR-10]; 108 [52-22; IFO 15832]; 143 [IFO 15836; PL]; 149 [2-40-40;
IFO
15838]; 182 [IFO 3081; PJ 73]; 184 [IFO 15830]; 185 [W2 L-1]; 186 [IFO 15829;
PJ
79]; 187 [NCPPB 263]; 188 [NCPPB 316]; 189 [PJ227; 1208]; 191 [TF'O 15834; PJ
236; 22/1]; 194 [Klinge R-60; PJ 253]; 196 [PJ 288]; 197 [PJ 290]; 198 [PJ
302]; 201
[PJ 368]; 202 [PJ 372]; 203 [PJ 376]; 204 [IFO 15835; PJ 682]; 205 [PJ 686];
206
[PJ 692]; 207 [PJ 693]; 208 [PJ 722]; 212 [PJ 832]; 215 [PJ 849]; 216 [PJ
885]; 267
[B-9]; 271 [B-1612]; 401 [C71A; IFO 15831; PJ 187]; NRRL B-3178 [4; IFO
15841]; KY 8521; 3081; 30-21; [IFO 3081]; N; PYR; PW; D946-B83 [BU 2183;
FERM-P 3328]; P-2563 [FERM-P 2894; IFO 13658]; IAM-1126 [43F]; M-1; A506
36


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
[AS-06]; A505 [AS-OS-1]; A526 [AS-26]; B69; 72; NRRL B-4290; PMW6 [NCIB
11615]; SC 12936; A1 [1F0 15839]; F 1847 [CDC-EB]; F 1848 [CDC 93]; NCIB
10586; P17; F-12; AmMS 257; PRA25; 6133D02; 6519E01; Nl; SC15208; BNL
WVC; NCTC 2583 [NC1B 8194]; H13; 1013 [ATCC 11251; CCEB 295]; IFO 3903;
1062; or Pf 5.
II. NUCLEIC ACID CONSTRUCTS
The present invention further provides nucleic acid constructs encoding a
fusion peptide of an icosahedral capsid and a recombinant peptide. In one
embodiment, a nucleic acid construct for use in transforming a Pseudomonad
host cell
including a) a nucleic acid encoding a recombinant peptide, and b) a nucleic
acid
sequence encoding an icosahedral capsid is provided, wherein the nucleic acid
of a)
and the nucleic acid of b) are operably linked to form a fusion protein when
expressed
in a cell.
In certain embodiments, the vector can include sequence for multiple capsids,
or for multiple peptides of interest. In one embodiment, the vector can
include at least
two different capsid-peptide coding sequences. In one embodiment, the coding
sequences are linked to the same promoter. In certain embodiments, the coding
sequences are separated by an internal ribosomal binding site. In other
embodiments,
the coding sequences are linked by a linker sequence that allows the formation
of
virus like particles in the cell. In another embodiment, the coding sequences
are
linked to different promoters. These promoters may be driven by the same
induction
conditions. In another embodiment, multiple vectors encoding different capsid-
peptide combinations are provided. The multiple vectors can include promoters
that
are driven by the same induction conditions, or by different induction
conditions. In
one embodiment, the promoter is a lac promoter, or a derivative of the lac
promoter
such as a tac promoter.
The coding sequence for a peptide of interest can be inserted into the coding
sequence for a viral capsid or capsid in a predetermined site. The peptide can
also be
inserted at a non-predetermined site and cells screened for production of
VLPs. In
one embodiment, the peptide is inserted into the capsid coding sequence so as
to be
expressed as a loop during formation of a VLP. In one embodiment, one peptide
coding sequence is included in the vector, however in other embodiments,
multiple
37


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
sequences are included. The multiple sequences can be in the form of
concatamers,
for example concatamers linked by cleavable linker sequences.
Peptides may be inserted at more than one insertion site in a capsid. Thus,
peptides may be inserted in more than one surface loop motif of a capsid;
peptides
may also be inserted at multiple sites within a given loop motif. The
individual
functional and/or structural peptides) of the insert(s), and/or the entire
peptide
insert(s), may be separated by cleavage sites, i.e. sites at which an agent
that cleaves
or hydrolyzes protein can act to separate the peptides) from the remainder of
the
capsid structure or assemblage.
Peptides may be inserted within external-facing loops) and/or within internal-
facing loop(s), i. e. within loops of the capsid that face respectively away
from or
toward the center of the capsid. Any amino acid or peptide bond in a surface
loop of a
capsid can serve as an insertion for the peptide. Typically, the insertion
site will be
selected at about the center of the loop, i.e. at about the position located
most distal
from the center of the tertiary structure of the folded capsid peptide. The
peptide
coding sequence may be operably inserted within the position of the capsid
coding
sequence corresponding to this approximate center of the selected loop(s).
This
includes the retention of the reading frame for that portion of the peptide
sequence of
the capsid that is synthesized downstream from the peptide insertion site.
In another embodiment, the peptide can be inserted at the amino terminus of
the capsid. The peptide can be linked to the capsid through one or more linker
sequences, including the cleavable linkers described above. In yet another
embodiment, the peptide can be inserted at the carboxy terminus of the capsid.
The
peptide can also be linked to the carboxy terminus through one or more
linkers, which
can be cleavable by chemical or enzymatic hydrolysis. In one embodiment,
peptide
sequences are linked at both the amino and carboxy termini, or at one terminus
and at
at least one internal location, such as a location that is expressed on the
surface of the
capsid in its three dimensional conformation.
In one embodiment, the peptide can be inserted into the capsid from a Cowpea
Chlorotic mosaic virus. In one particular embodiment, the peptide can be
inserted at
amino acid 129 of the CCMV virus. In another embodiment, the peptide sequence
can be inserted at amino acids 60, 61, 62 or 63 of the CCMV virus. In still
another
embodiment, the peptide can be inserted at both amino acids 129 and amino
acids 60-
63 of the CCMV virus.
38


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
In a particular embodiment, the present invention provides a nucleic acid
construct including a) a nucleic acid encoding an antimicrobial peptide, and
b) a
nucleic acid encoding an icosahedral capsid, wherein the nucleic acid of a)
and the
nucleic acid of b) are operably linked to form a fusion protein when expressed
in a
cell. Other capsids and recombinant peptides useful in constructing the
nucleic acid
construct are disclosed above.
Promotes
In one embodiment, the nucleic acid construct includes a promoter sequence
operably attached to the nucleic acid sequence encoding the capsid-recombinant
peptide fusion peptide. An operable attachment or linkage refers to any
configuration
in which the transcriptional and any translational regulatory elements are
covalently
attached to the described sequence so that by action of the host cell, the
regulatory
elements can direct the expression of the sequence of interest.
In a fermentation process, once expression of the target recombinant peptide
is
induced, it is ideal to have a high level of production in order to maximize
efficiency
of the expression system. The promoter initiates transcription and is
generally
positioned 10-100 nucleotides upstream of the ribosome binding site. Ideally,
a
promoter will be strong enough to allow for recombinant peptide accumulation
of
around 50% of the total cellular protein of the host cell, subject to tight
regulation,
and easily (and inexpensively) induced.
The promoters used in accordance with the present invention may be
constitutive promoters or regulated promoters. Examples of commonly used
inducible promoters and their subsequent inducers include lac (IPTG), lacUVS
(IPTG), tac (IPTG), trc (IPTG), PSS," (IPTG), trp (tryptophan starvation),
araBAD (1-
arabinose), lppa (IPTG), lpp-lac (IPTG), phoA (phosphate starvation), recA
(nalidixic
acid), proU (osmolarity), cst-1 (glucose starvation), tetA (tretracylin), cadA
(pH), nar
(anaerobic conditions), PL (thermal shift to 42° C), cspA (thermal
shift to 20° C), T7
(thermal induction), T7-lac operator (1PTG), T3-lac operator (IPTG), TS-lac
operator
(IPTG), T4 gene32 (T4 infection), nprM-lac operator (IPTG), Pm (alkyl- or halo-

benzoates), Pu (alkyl- or halo-toluenes), Psal (salicylates), and VHb
(oxygen). See,
for example, Makrides, S.C. (1996) Microbiol. Rev. 60, 512-538; Hannig G. &
Makrides, S.C. (1998) TIBTECH 16, 54-60; Stevens, R.C. (2000) Structures 8,
R177-
8185. See, e.g.: J. Sanchez-Romero & V. De Lorenzo, Genetic Engineering of
39


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
Nonpathogenic Pseudonzonas strains as Biocatalysts for Industrial and
Environmental
Processes, in Manual of Industrial Microbiology and Biotechnology (A. Demain ~
J.
Davies, eds.) pp.460-74 (1999) (ASM Press, Washington, D.C.); H. Schweizer,
Vectors to express foreign genes and techniques to monitor gene expression for
Pseudomonads, Current Opinion in Biotechnology, 12:439-445 (2001); and R.
Slater
~ R. Williams, The Expression of Foreign DNA in Bacteria, in Molecular Biology
and Biotechnology (J. Walker & R. Rapley, eds.) pp.125-54 (2000) (The Royal
Society of Chemistry, Cambridge, UI~).
A promoter having the nucleotide sequence of a promoter native to the
selected bacterial host cell can also be used to control expression of the
transgene
encoding the target peptide, e.g., a Pseudofnonas anthranilate or benzoate
operon
promoter (Pant, Pben). Tandem promoters may also be used in which more than
one
promoter is covalently attached to another, whether the same or different in
sequence,
e.g., a Pant-Pben tandem promoter (interpromoter hybrid) or a Plac-Plac tandem
promoter.
Regulated promoters utilize promoter regulatory proteins in order to control
transcription of the gene of which the promoter is a part. Where a regulated
promoter
is used herein, a corresponding promoter regulatory protein will also be part
of an
expression system according to the present invention. Examples of promoter
regulatory proteins include: activator proteins, e.g., E. coli catabolite
activator protein,
MaIT protein; AraC family transcriptional activators; repressor proteins,
e.g., E. coli
LacI proteins; and dual-faction regulatory proteins, e.g., E. coli NagC
protein. Many
regulated-promoter/promoter-regulatory-protein pairs are known in the art.
Promoter regulatory proteins interact with an effector compound, i.e. a
compound that reversibly or irreversibly associates with the regulatory
protein so as to
enable the protein to either release or bind to at least one DNA transcription
regulatory region of the gene that is under the control of the promoter,
thereby
permitting or blocking the action of a transcriptase enzyme in initiating
transcription
of the gene. Effector compounds are classified as either inducers or co-
repressors,
and these compounds include native effector compounds and gratuitous inducer
compounds. Many regulated-promoter/promoter-regulatory-protein/effector-
compound trios are known in the art. Although an effector compound can be used
throughout the cell culture or fermentation, in a particular embodiment in
which a
regulated promoter is used, after growth of a desired quantity or density of
host cell


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
biomass, an appropriate effector compound is added to the culture in order to
directly
or indirectly result in expression of the desired target gene(s).
By way of example, where a lac family promoter is utilized, a lacl gene, or
derivative thereof such as a laclQ or laclQl gene, can also be present in the
system.
The lacl gene, which is (normally) a constitutively expressed gene, encodes
the Lac
repressor protein (LacI protein) which binds to the lac operator of these
promoters.
Thus, where a lac family promoter is utilized, the lacl gene can also be
included and
expressed in the expression system. In the case of the lac promoter family
members,
e.g., the tac promoter, the effector compound is an inducer, preferably a
gratuitous
inducer such as IPTG (isopropyl-(3-D-1-thiogalactopyranoside, also called
"isopropylthiogalactoside").
In a particular embodiment, a lac or tac family promoter is utilized in the
present invention, including Plac, Ptac, Ptrc, PtacII, PlacUVS, lpp-PIacUVS,
lpp-lac,
nprM-lac, T7lac, TSlac, T3lac, and Pmac.
Other Elemehts
Other regulatory elements can be included in an expression construct,
including lac0 sequences. Such elements include, but are not limited to, for
example,
transcriptional enhancer sequences, translational enhancer sequences, other
promoters,
activators, translational start and stop signals, transcription terminators,
cistronic
regulators, polycistronic regulators, tag sequences, such as nucleotide
sequence "tags"
and "tag" peptide coding sequences, which facilitates identification,
separation,
purification, or isolation of an expressed peptide, including His-tag, Flag-
tag, T7-tag,
S-tag, HSV-tag, B-tag, Strep-tag, polyarginine, polycysteine,
polyphenylalanine,
polyaspartic acid, (Ala-Trp-Trp-Pro)n, thioredoxin, beta-galactosidase,
chloramphenicol acetyltransferase, cyclomaltodextrin gluconotransferase,
CTP:CMP-
3-deoxy-D-manno-octulosonate cytidyltransferase, trpE or trpLE, avidin,
streptavidin,
T7 gene 10, T4 gp55, Staphylococcal protein A, streptococcal protein G, GST,
DHFR,
CBP, MBP, galactose binding domain, Calinodulin binding domain, GFP, KSI, c-
myc,
ompT, ompA, pelB, , NusA, ubiquitin, and hemosylin A.
In one embodiment, the nucleic acid construct further comprises a tag
sequence adjacent to the coding sequence for the recombinant peptide of
interest, or
linked to a coding sequence for a viral capsid. In one embodiment, this tag
sequence
41


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
allows for purification of the protein. The tag sequence can be an affinity
tag, such as
a hexa-histidine affinity tag. In another embodiment, the affinity tag can be
a
glutathione-S-transferase molecule. The tag can also be a fluorescent
molecule, such
as YFP or GFP, or analogs of such fluorescent proteins. The tag can also be a
portion
of an antibody molecule, or a known antigen or ligand for a known binding
partner
useful for purification.
The present invention can include, in addition to the capsid-recombinant
peptide coding sequence, the following regulatory elements operably linked
thereto: a
promoter, a ribosome binding site (RBS), a transcription terminator,
translational start
and stop signals. Useful RBSs can be obtained from any of the species useful
as host
cells in expression systems according to the present invention, preferably
from the
selected host cell. Many specific and a variety of consensus RBSs are known,
e.g.,
those described in and referenced by D. Frishman et al., Starts of bacterial
genes:
estimating the reliability of computer predictions, Gene 234(2):257-65 (8 Jul
1999);
and B.E. Suzek et al., A probabilistic method for identifying start codons in
bacterial
genomes, Bioinformatics 17(12):1123-30 (Dec 2001). In addition, either native
or
synthetic RBSs may be used, e.g., those described in: EP 0207459 (synthetic
RBSs);
O. Ikehata et al., Primary structure of nitrite hydratase deduced from the
nucleotide
sequence of a Rhodococcus species and its expression in Escherichia coli, Eur.
J.
Biochem. 181(3):563-70 (1989) (native RBS sequence of AAGGAAG). Further
examples of methods, vectors, and translation and transcription elements, and
other
elements useful in the present invention are described in, e.g.: US Patent No.
5,055,294 to Gilroy and US Patent No. 5,128,130 to Gilroy et al.; US Patent
No.
5,281,532 to Rammler et al.; US Patent Nos. 4,695,455 and 4,861,595 to Barnes
et al.;
US Patent No. 4,755,465 to Gray et al.; and US Patent No. 5,169,760 to Wilcox.
T~ecto~s
Transcription of the DNA encoding the enzymes of the present invention by a
Pseudomonad host can further be increased by inserting an enhancer sequence
into the
vector or plasmid. Typical enhancers are cis-acting elements of DNA, usually
about
from 10 to 300 by in size that act on the promoter to increase its
transcription.
Generally, the recombinant expression vectors will include origins of
replication and selectable markers permitting transformation of the
Pseudomonad host
cell, e.g., the capsid-recombinant peptide fusion peptides of the present
invention, and
42


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
a promoter derived from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters have been described above. The
heterologous structural sequence is assembled in appropriate phase with
translation
initiation and termination sequences. Optionally, and in accordance with the
present
invention, the heterologous sequence can encode a fusion peptide including an
N-
terminal identification peptide imparting desired characteristics, e.g.,
stabilization or
simplified purification of expressed recombinant product.
Useful expression vectors for use with P. fluorescehs in expressing capsid
recombinant peptide fusion peptides are constructed by inserting a structural
DNA
sequence encoding a desired target peptide fused with a capsid peptide
together with
suitable translation initiation and termination signals in operable reading
phase with a
functional promoter. The vector will comprise one or more phenotypic
selectable
markers and an origin of replication to ensure maintenance of the vector and
to, if
desirable, provide amplification within the host. Suitable hosts for
transformation in
accordance with the present disclosure include various species within the
genera
Pseudomohas, and, in particular, the host cell strain of Pseudomonas
fluo~escefzs.
Vectors are known in the art as useful for expressing recombinant proteins in
host cells, and any of these may be modified and used for expressing the
fusion
products according to the present invention. Such vectors include, e.g.,
plasmids,
cosmids, and phage expression vectors. Examples of useful plasmid vectors that
can
be modified for use on the present invention include, but are not limited to,
the
expression plasmids pBBRIMCS, pDSK519, pKT240, pML122, pPSlO, RK2, RK6,
pR01600, and RSF1010. Further examples can include PALTER-Exl, PALTER-Ex2,
pBAD/His, pBAD/Myc-His, pBAD/gIII, pCal-n, pCal-n-EK, pCal-c, pCal-Kc,
pcDNA 2.1, pDUAL, PET-3a-c, PET 9a-d, PET-11a-d, PET-12a-c, PET-14b, pETlSb,
PET-16b, PET-17b, PET-19b, PET-20b(+), PET-21a-d(+), PET-22b(+), PET-23a-d(+),
pET24a-d(+), PET-25b(+), PET-26b(+), PET-27b(+), pET28a-c(+), PET-29a-c(+),
PET-30a-c(+), pET3lb(+), PET-32a-c(+), PET-33b(+), PET-34b(+), pET35b(+), pET-
36b(+), PET-37b(+), PET-38b(+), PET-39b(+), PET-40b(+), PET-41a-c(+), PET-42a-
c(+), PET-43a-c(+), pETBlue-1, pETBlue-2, pETBlue-3, pGEMEX-1, pGEMEX-2,
pGEXI~,T, pGEX-2T, pGEX-2TK, pGEX-3X, pGEX-4T, pGEX-SX, pGEX-6P,
pHATlO/11/12, pHAT20, pHAT-GFPuv, pKK223-3, ALEX, pMAL-c2X, pMAL-c2E,
pMAL-c2g, pMAL-p2X, pMAL-p2E, pMAL-p2G, pProEX HT, pPROLar.A,
pPROTet.E, pQE-9, pQE-16, pQE-30/31/32, pQE-40, pQE-50, pQE-70, pQE-
43


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
80/81/82L, pQE-100, pRSET, and pSE280, pSE380, pSE420, pThioHis, pTrc99A,
pTrcHis, pTrcHis2, pTriEx-1, pTriEx-2, pTrxFus. Other examples of such useful
vectors include those described by, e.g.: N. Hayase, in Appl. Envir.
Microbiol.
60(9):3336-42 (Sep 1994); A.A. Lushnikov et al., in Basic Life Sci. 30:657-62
(1985); S. Graupner & W. Wackernagel, in Biomolec. Eng. 17(1):11-16. (Oct
2000);
H.P. Schweizer, in Curr. Opin. Biotech. 12(5):439-45 (Oct 2001); M.
Bagdasarian &
K.N. Timmis, in Curr. Topics Microbiol. Immunol. 96:47-67 (1982); T. Ishii et
al., in
FEMS Microbiol. Lett. 116(3):307-13 (Mar 1, 1994); LN. Olekhnovich ~. Y.K.
Fomichev, in Gene 140(1):63-65 (Mar 11, 1994); M. Tsuda & T. Nakazawa, in Gene
136(1-2):257-62 (Dec 22, 1993); C. Nieto et al., in Gene 87(1):145-49 (Mar 1,
1990);
J.D. Jones & N. Gutterson, in Gene 61(3):299-306 (1987); M. Bagdasarian et
al., in
Gene 16(1-3):237-47 (Dec 1981); H.P. Schweizer et al., in Genet. Eng. (NY)
23:69-
81 (2001); P. Mukhopadhyay et al., in J. Bact. 172(1):477-80 (Jan 1990); D.O.
Wood
et al., in J. Bact. 145(3):1448-51 (Mar 1981); and R. Holtwick et al., in
Microbiology
147(Pt 2):337-44 (Feb 2001).
Further examples of expression vectors that can be useful in Pseudornonas
host cells include those listed in Table 2 as derived from the indicated
replicons.
TABLE 2. SOME EXAMPLES OF USEFUL EXPRESSION VECTORS
Replicon Vectors)


pPSlO pCN39, pCN51


RSF1010 pKT261-3


pMMB66EH


pEBB


pPLGNI


pMYCl 050


RK2/RP 1 pRK415


pJB653


pRO1600 pUCP


pBSP


The expression plasmid, RSF1010, is described, e.g., by F. Heffron et al., in
Proc. Nat'1 Acad. Sci. USA 72(9):3623-27 (Sep 1975), and by K. Nagahari & K.
44


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
Sakaguchi, in J. Bact. 133(3):1527-29 (Mar 1978). Plasmid RSF1010 and
derivatives
thereof are particularly useful vectors in the present invention. Exemplary,
useful
derivatives of RSF1010, which are l~nown in the art, include, e.g., pKT212,
pKT214,
pKT231 and related plasmids, and pMYC1050 and related plasmids (see, e.g., US
Patent Nos. 5,527,883 and 5,840,554 to Thompson et al.), such as, e.g.,
pMYC1803.
Plasmid pMYC1803 is derived from the RSF1010-based plasmid pTJS260 (see US
Patent No. 5,169,760 to Wilcox), which carries a regulated tetracycline
resistance
marker and the replication and mobilization loci from the RSF1010 plasmid.
Other
exemplary useful vectors include those described in US Patent No. 4,680,264 to
Puhler et al.
In a one embodiment, an expression plasmid is used as the expression vector.
In another embodiment, RSF1010 or a derivative thereof is used as the
expression
vector. In still another embodiment, pMYC1050 or a derivative thereof, or
pMYC1803 or a derivative thereof, is used as the expression vector.
The ChampionTM pET expression system provides a high level of protein
production. Expression is induced from the strong T7lac promoter. This system
takes
advantage of the high activity and specificity of the bacteriophage T7 RNA
polymerase for high level transcription of the gene of interest. The lac
operator
located in the promoter region provides tighter regulation than traditional T7-
based
vectors, improving plasmid stability and cell viability (Studier, F. W. and B.
A.
Moffatt (1986) J Molecular Biology 189(1): 113-30; Rosenberg, et al. (1987)
Gene
56(1): 125-35). The T7 expression system uses the T7 promoter and T7 RNA
polymerase (T7 RNAP) for high-level transcription of the gene of interest.
High-level
expression is achieved in T7 expression systems because the T7 RNAP is more
processive than native E. coli RNAP and is dedicated to the transcription of
the gene
of interest. Expression of the identified gene is induced by providing a
source of T7
RNAP in the host cell. This is accomplished by using a BL21 E. coli host
containing a
chromosomal copy of the T7 RNAP gene. The T7 RNAP gene is under the control of
the lacUVS promoter which can be induced by IPTG. T7 RNAP is expressed upon
induction and transcribes the gene of interest.
The pBAD expression system allows tightly controlled, titratable expression
of recombinant protein through the presence of specific carbon sources such as
glucose, glycerol and arabinose (Guzman, et al. (1995) JBacteriology 177(14):
4121-
30). The pBAD vectors are uniquely designed to give precise control over
expression


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
levels. Heterologous gene expression from the pBAD vectors is initiated at the
a~aBAD promoter. The promoter is both positively and negatively regulated by
the
product of the araC gene. AraC is a transcriptional regulator that forms a
complex
with L-arabinose. In the absence of L-arabinose, the AraC dimer blocks
transcription.
For maximum transcriptional activation two events are required: (i) L-
arabinose binds
to AraC allowing transcription to begin. (ii.) The cAMP activator protein
(CAP)-
cAMP complex binds to the DNA and stimulates binding of AraC to the correct
location of the promoter region.
The trc expression system allows high-level, regulated expression in E. coli
from the tYC promoter. The tie expression vectors have been optimized for
expression
of eukaryotic genes in E. coli. The tic promoter is a strong hybrid promoter
derived
from the tryptophane (trp) and lactose (lac) promoters. It is regulated by the
lac~
operator and the product of the lacIQ gene (Brosius, J. (1984) Gene 27(2): 161-
72).
III. EXPRESSION OF VIRUS LIKE PARTICLES IN PSEUDOMONADS
The present invention also provides a process for producing a recombinant
peptide. The process includes:
a) providing a Pseudomonad cell;
b) providing a nucleic acid encoding a fusion peptide; wherein the fusion is
of a recombinant peptide and an icosahedral capsid;
c) expressing the nucleic acid in the Pseudomonad cell, wherein the
expression in the cell provides for in vivo assembly of the fusion peptide
into virus like particles; and
d) isolating the virus like particles.
Peptides may be expressed as single-copy peptide inserts within a capsid
peptide (i.e. expressed as individual inserts from recombinant capsid peptide
coding
sequences that are mono-cistronic for the peptide) or may be expressed as di-,
tri-, or
multi-copy peptide inserts (i.e. expressed as concatemeric inserts from
recombinant
capsid peptide coding sequences that are poly-cistronic for the peptide; the
concatemeric inserts) may contain multiple copies of the same exogenous
peptide of
interest or may contain copies of different exogenous peptides of interest).
Concatemers may be homo- or hetero-concatemers.
46


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
In one embodiment, the isolated virus like particle can be administered to a
human or animal in a vaccine strategy.
In another embodiment, the nucleic acid construct can be co-expressed with
another nucleic acid encoding a wild type capsid. In a particular embodiment,
the co
y expressed capsid/capsid-recombinant peptide fusion particles assemble in
vivo to form
a chimeric virus like particle. The chimeric VLP is a virus like particle
including
capsids or capsid-peptide fusions encoded by at least two different nucleic
acid
constructs.
In still another embodiment, the nucleic acid construct can be co-expressed
with another nucleic acid encoding a different capsid-recombinant peptide
fusion
particle. In a particular embodiment, the co-expressed capsid fusion particles
will
assemble in vivo to form a chimeric virus like particle.
In still another embodiment, a second nucleic acid, which is designed to
express a different peptide, such as a chaperone protein, can be expressed
concomitantly with the nucleic acid encoding the fusion peptide.
The Pseudomonad cells, capsids, and recombinant peptides useful for the
present invention are discussed above.
In one embodiment, the process produces at least 0.1 g/L protein in the form
of VLPs. In another embodiment, the process produces 0.1 to 10 g/L protein in
the
form of VLPs. In subembodiments, the process produces at least about 0.2, 0.3,
0.4,
0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 g/L protein in the form of VLPs or cage
structures. In one
embodiment, the total recombinant protein produced is at least 1.0 g/L. In
some
embodiments, the amount of VLP protein produced is at least about 5%, about
10%,
about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%,
about 70%, about 80%, about 90%, about 95% or more of total recombinant
protein
produced.
In one embodiment, the process produces at least 0.1 g/L pre-formed VLPs or
cage structures. In another embodiment, the process produces 0.1 to 10 g/L pre-

formed VLPs in the cell. In another embodiment, the process produces 0.1 to 10
g/L
pre-formed cage structures in the cell. In subembodiments, the process
produces at
least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 g/L pre-formed VLPs.
In one
embodiment, the total pre-formed VLP protein produced is at least 1.0 g/L. hl
subembodiments, the total VLP protein produced can be at least about 2.0, 3.0,
4.0,
5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 15.0, 20.0 or 50.0 g/L. In some embodiments,
the amount
47


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
of VLP protein produced is at least about 5%, about 10%, about 15%, about 20%,
about 25%, or more of total recombinant protein produced.
In another embodiment, more than 50% of the expressed, transgenic peptide,
peptide, protein, or fragment thereof produced can be produced in a
renaturable form
in host cell. In another embodiment about 60%, 70%, 75%, 80%, 85%, 90%, 95% of
the expressed protein is obtained in or can be renatured into active form.
The process of the invention can also lead to increased yield of recombinant
protein. In one embodiment, the process produces recombinant protein as 5, 10,
15,
20, 25, 30, 40 or 50, 55, 60, 65, 70, or 75 % of total cell protein (tcp).
"Percent total
cell protein " is the amount of peptide in the host cell as a percentage of
aggregate
cellular protein. The determination of the percent total cell protein is well
known in
the art.
In a particular embodiment, the host cell can have a recombinant peptide,
peptide, protein, or fragment thereof expression level of at least 1 % tcp and
a cell
density of at least 40 g/L, when grown (i.e. within a temperature range of
about 4° C
to about 55° C, inclusive) in a mineral salts medium. In a particular
embodiment, the
expression system will have a recombinant protein of peptide expression level
of at
least 5% tcp and a cell density of at least 40 glL, when grown (i.e. within a
temperature range of about 4° C to about 55° C, inclusive) in a
mineral salts medium
at a fermentation scale of at least 10 Liters.
In a separate embodiment, a portion of the expressed viral capsid operably
linked to a peptide of interest is formed in an insoluble aggregate in the
cell. In one
embodiment, the peptide of interest can be renatured from the insoluble
aggregate.
Cleavage of Peptide of IfZtef°est
In one embodiment, the process further provides: e) cleaving the fusion
product to separate the recombinant peptide from the capsid.
A cleavable linkage sequence can be included between the viral protein and
the recombinant peptide. Examples of agents that can cleave such sequences
include,
but are not limited to chemical reagents such as acids (HCl, formic acid),
CNBr,
hydroxylamine (for asparagine-glycine), 2-Nitro-5- thiocyanobenzoate, O-
Iodosobenzoate, and enzymatic agents, such as endopeptidases, endoproteases,
trypsin,
clostripain, and Staphylococcal protease.
48


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
Cleavable linkage sequences are well known in the art. In the present
invention, any cleavable linkage sequence recognized by cleavage agents,
including
dipeptide cleavage sequences such as Asp-Pro, can be utilized.
Expression
The process of the invention optimally leads to increased production of
recombinant peptide in a host cell. The increased production alternatively can
be an
increased level of active peptide per gram of protein produced, or per gram of
host
protein. The increased production can also be an increased level of
recoverable
peptide, such as soluble protein, produced per gram of recombinant or per gram
of
host cell protein. The increased production can also be any combination of
increased
total level and increased active or soluble level of protein.
The improved expression of recombinant protein can be through expression of
the protein inserted in VLPs. In certain embodiments, at least 60, at least
70, at least
80, at least 90, at least 100, at least 110, at least 120, at least 130, at
least 140, at least
150, at least 160, at least 170, or at least 180 copies of a peptide of
interest is
expressed in each VLP. The VLPs can be produced and recovered from the
cytoplasm, periplasm or extracellular medium of the host cell.
In another embodiment, the peptide can be insoluble in the cell. In certain
embodiments, the insoluble peptide is produced in a particle formed of
multiple
capsids but not forming a native-type VLP. For example, a cage structure of as
few as
3 viral capsids can be formed. In certain embodiments, the capsid structure
includes
more than one copy of a peptide of interest and in certain embodiments,
includes at
least ten, at least 20, or at least 30 copies.
The peptide or viral capsid sequence can also include one or more targeting
sequences or sequences to assist purification. These can be an affinity tag.
These
can also be targeting sequences directing the assembly of capsids into a VLP.
Cell Growth
Transformation of the Pseudonaofzas host cells with the vectors) may be
performed using any transformation methodology known in the art, and the
bacterial
host cells may be transformed as intact cells or as protoplasts (i.e.
including
cytoplasts). Exemplary transformation methodologies include poration
methodologies, e.g., electroporation, protoplast fusion, bacterial
conjugation, and
divalent cation treatment, e.g., calcium chloride treatment or CaCI/Mg2+
treatment, or
49


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
other well known methods in the art. See, e.g., Morrison, J. Bact., 132:349-
351
(1977); Clark-Curtiss ~ Curtiss, Methods in Enzymology, 101:347-362 (Wu et
al.,
eds, 1983), Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed.
1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and
Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).
As used herein, the term "fermentation" includes both embodiments in which
literal fermentation is employed and embodiments in which other, non-
fermentative
culture modes are employed. Fermentation may be performed at any scale. In one
embodiment, the fermentation medium may be selected from among rich media,
minimal media, and mineral salts media; a rich medium may be used, but is
preferably
avoided. In another embodiment either a minimal medium or a mineral salts
medium
is selected.
In still another embodiment, a minimal medium is selected. In yet another
embodiment, a mineral salts medium is selected. Mineral salts media are
particularly
preferred.
Mineral salts media consists of mineral salts and a carbon source such as,
e.g.,
glucose, sucrose, or glycerol. Examples of mineral salts media include, e.g.,
M9
medium, Pseudomohas medium (ATCC 179), Davis and Mingioli medium (see, BD
Davis & ES Mingioli (1950) in J. Bact. 60:17-28). The mineral salts used to
make
mineral salts media include those selected from among, e.g., potassium
phosphates,
ammonium sulfate or chloride, magnesium sulfate or chloride, and trace
minerals
such as calcium chloride, borate, and sulfates of iron, copper, manganese, and
zinc.
No organic nitrogen source, such as peptone, tryptone, amino acids, or a yeast
extract,
is included in a mineral salts medium. Instead, an inorganic nitrogen source
is used
and this may be selected from among, e.g., ammonium salts, aqueous ammonia,
and
gaseous ammonia. A preferred mineral salts medium will contain glucose as the
carbon source. In comparison to mineral salts media, minimal media can also
contain
mineral salts and a carbon source, but can be supplemented with, e.g., low
levels of
amino acids, vitamins, peptones, or other ingredients, though these are added
at very
minimal levels.
The high cell density culture can start as a batch process which is followed
by
a two-phase fed-batch cultivation. After unlimited growth in the batch part,
growth
can be controlled at a reduced specific growth rate over a period of 3
doubling times
in which the biomass concentration can increased several fold. Further details
of such


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
cultivation procedures is described by Riesenberg, D.; Schulz, V.; Knorre, W.
A.;
Pohl, H. D.; Korz, D.; Sanders, E. A.; Ross, A.; Deckwer, W. D. (1991) "High
cell
density cultivation of Escherichia coli at controlled specific growth rate" J
Biotechnol: 20(1) 17-27.
The expression system according to the present invention can be cultured in
any fermentation format. For example, batch, fed-batch, semi-continuous, and
continuous fermentation modes may be employed herein.
The expression systems according to the present invention are useful for
transgene expression at any scale (i.e. volume) of fermentation. Thus, e.g.,
microliter-
scale, centiliter scale, and deciliter scale fermentation volumes may be used;
and 1
Liter scale and larger fermentation volumes can be used. In one embodiment,
the
fermentation volume will be at or above 1 Liter. In another embodiment, the
fermentation volume will be at or above 5 Liters, 10 Liters, 15 Liters, 20
Liters, 25
Liters, 50 Liters, 75 Liters, 100 Liters, 200 Liters, 500 Liters, 1,000
Liters, 2,000
Liters, 5,000 Liters, 10,000Liters or 50,000 Liters.
In the present invention, growth, culturing, and/or fermentation of the
transformed host cells is performed within a temperature range permitting
survival of
the host cells, preferably a temperature within the range of about 4°C
to about 55°C,
inclusive. Thus, e.g., the terms "growth" (and "grow," "growing"), "culturing"
(and
"culture"), and "fermentation" (and "ferment," "fermenting"), as used herein
in regaxd
to the host cells of the present invention, inherently means "growth,"
"culturing," and
"fermentation," within a temperature range of about 4°C to about
55°C, inclusive. In
addition, "growth" is used to indicate both biological states of active cell
division
and/or enlargement, as well as biological states in which a non-dividing
and/or non-
enlarging cell is being metabolically sustained, the latter use of the term
"growth"
being synonymous with the term "maintenance."
Cell Density
An additional advantage in using Pseudomonas fluo~escefzs in expressing
recombinant peptides encased in VLPs includes the ability of Pseudomofaas
fluorescefas to be grown in high cell densities compared to E. coli or other
bacterial
expression systems. To this end, Pseudofraonas fluo~esceras expressions
systems
according to the present invention can provide a cell density of about 20 g/L
or more.
The Pseudonzofzas fZuorescens expressions systems according to the present
invention
51


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
can likewise provide a cell density of at least about 70 g/L, as stated in
terms of
biomass per volume, the biomass being measured as dry cell weight.
In one embodiment, the cell density will be at least 20 g/L. In another
embodiment, the cell density will be at least 25 g/L, 30 g/L, 35 g/L, 40 g/L,
45 g/L, 50
g/L, 60 g/L, 70 g/L, 80 g/L, 90 g/L., 100 g/L, 110 g/L, 120 g/L, 130 g/L, 140
g/L, or
at least 150 g/L.
In another embodiments, the cell density at induction will be between 2 0 g/L
and 150 g/L;, 20 g/L and 120 g/L; 20 g/L and 80 g/L; 25 g/L and 80 g/L; 30 g/L
and
80 g/L; 35 g/L and 80 g/L; 40 g/L and 80 g/L; 45 g/L and 80 g/L; 50 g/L and 80
g/L;
50 g/L and 75 g/L; 50 g/L and 70 g/L; 40 g/L and 80 g/L.
Isolation of hLP oY Peptide of IfZteYest
In certain embodiments, the invention provides a process for improving the
recovery of peptides of interest by protection of the peptide during
expression through
linkage and co-expression with a viral capsid. In certain embodiments, the
viral
capsid fusion forms a VLP, which can be readily separated from the cell
lysate.
The proteins of this invention may be isolated purified to substantial purity
by
standard techniques well known in the art, including, but not limited to,
ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, nickel chromatography,
hydroxylapatite
chromatography, reverse phase chromatography, lectin chromatography,
preparative
electrophoresis, detergent solubilization, selective precipitation with such
substances
as column chromatography, immunopurification methods, and others. For example,
proteins having established molecular adhesion properties can be reversibly
fused a
ligand. With the appropriate ligand, the protein can be selectively adsorbed
to a
purification column and then freed from the column in a relatively pure form.
The
fused protein is then removed by enzymatic activity. In addition, protein can
be
purified using immunoaffinity columns or Ni-NTA columns. General techniques
axe
further described in, for example, R. Scopes, Protein Purification: Principles
and
Practice, Springer-Verlag: N.Y. (1982); Deutscher, Guide to Protein
Purification,
Academic Press (1990); U.S. Pat. No. 4,511,503; S. Roe, Protein Purification
Techniques: A Practical Approach (Practical Approach Series), Oxford Press
(2001);
D. Bollag, et al., Protein Methods, Wiley-Lisa, Inc. (1996); AK Patra et al.,
Protein
52


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
Expr Purif, 18(2): p/ 182-92 (2000); and R. Mukhija, et al., Gene 165(2): p.
303-6
(1995). See also, for example, Ausubel, et al. (1987 and periodic
supplements);
Deutscher (1990) "Guide to Protein Purification," Methods in Enzymology vol.
182,
and other volumes in this series; Coligan, et al. (1996 and periodic
Supplements)
Current Protocols in Protein Science Wiley/Greene, NY; and manufacturer's
literature
on use of protein purification products, e.g., Pharmacia, Piscataway, N.J., or
Bio-Rad,
Richmond, Calif. Combination with recombinant techniques allow fusion to
appropriate segments, e.g., to a FLAG sequence or an equivalent which can be
fused
via a protease-removable sequence. See also, for example., Hochuli (1989)
Chemische Industrie 12:69-70; Hochuli (1990) "Purification of Recombinant
Proteins
with Metal Chelate Absorbent" in Setlow (ed.) Genetic Engineering, Principle
and
Methods 12:87-98, Plenum Press, NY; and Crowe, et al. (1992) QIAexpress: The
High Level Expression & Protein Purification System QUIAGEN, Inc., Chatsworth,
Calif.
Similarly, the virus-like particles or cage-like structures can be isolated
and./or purified to substantial purity by standard techniques well known in
the art.
Techniques for isolation of VLPs, include, in addition to those described
above,
precipitation techniques such as polyethylene glycol or salt precitipation.
Separation
techniques include anion or cation exchange chromatography, size exclusion
chromatograph, phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, nickel chromatography,
hydroxylapatite
chromatography, reverse phase chromatography, lectin chromatography,
preparative
electrophoresis, immunopurification methods, centrifugation,
ultracentrifugation,
density gradient centrifugation (for example, on a sucrose or on a cesium
chloride
(CsCI) gradient), ultrafiltration through a size exclusion filter, and any
other protein
isolation methods known in the art.
The invention can also improve recovery of active recombinant peptides.
Levels of active protein can be measured, for example, by measuring the
interaction
between an identified and a parent peptide, peptide variant, segment-
substituted
peptide and/or residue-substituted peptide by any convenient ih vitro or ira
vivo assay.
Thus, ifa vitro assays can be used to determine any detectable interaction
between an
identified protein and a peptide of interest, e.g. between enzyme and
substrate,
between hormone and hormone receptor, between antibody and antigen, etc. Such
detection can include the measurement of colorimetric changes, changes in
53


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
radioactivity, changes in solubility, changes in molecular weight as measured
by gel
electrophoresis and/or gel exclusion processes, etc. In vivo assays include,
but are not
limited to, assays to detect physiological effects, e.g. weight gain, change
in
electrolyte balance, change in blood clotting time, changes in clot
dissolution and the
induction of antigenic response. Generally, any in vivo assay can be used so
long as a
variable parameter exists so as to detect a change in the interaction between
the
identified and the peptide of interest. See, for example, U.S. Patent No.
5,834,250.
To release recombinant proteins from the periplasm, treatments involving
chemicals such as chloroform (Ames et al. (1984) J. Bactef°iol., 160:
1181-1183),
guanidine-HCI, and Triton X-100 (Naglak and Wang (1990) Enzyme Micf°ob.
Teclanol., 12: 603-611) have been used. However, these chemicals are not inert
and
may have detrimental effects on many recombinant protein products or
subsequent
purification procedures. Glycine treatment of E. coli cells, causing
permeabilization
of the outer membrane, has also been reported to release the periplasmic
contents
(Ariga et al. (1989) J. Ferm. Bioeng., 68: 243-246). The most widely used
methods
of periplasmic release of recombinant protein are osmotic shock (Nasal and
Heppel
(1966) J. Biol. Chem., 241: 3055-3062; Neu and Heppel (1965) J. Biol. Claem.,
240:
3685-3692), hen egg white (HEW)-lysozyrne/ethylenediamine tetraacetic acid
(EDTA) treatment (Neu and Heppel (1964) J. Biol. Chem., 239: 3893-3900;
Witholt
et al. (1976) Biochim. Biophys. Acta, 443: 534-544; Pierce et al. (1995)
ICheme
Research. Event, 2: 995-997), and combined HEW-lysozyme/osmotic shock
treatment
(French et al. (1996) Enzyme and Micr~ob. Tech., 19: 332-338). The French
method
involves resuspension of the cells in a fractionation buffer followed by
recovery of the
periplasmic fraction, where osmotic shock immediately follows lysozyrne
treatment.
The effects of overexpression of the recombinant protein, S. thermoviolaceus a-

amylase, and the growth phase of the host organism on the recovery are also
discussed.
Typically, these procedures include an initial disruption in osmotically-
stabilizing medium followed by selective release in non-stabilizing medium.
The
composition of these media (pH, protective agent) and the disruption methods
used
(chloroform, HEW-lysozyme, EDTA, sonication) vary among specific procedures
reported. A variation on the HEW-lysozyme/EDTA treatment using a dipolar ionic
detergent in place of EDTA is discussed by Stabel et al. (1994) Yeterina~y
MicYObiol.,
38: 307-314. For a general review of use of intracellular lytic enzyme systems
to
54


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
disrupt E. coli, see Dabora and Cooney (1990 )in Advances in Biochemical
Engineering/Biotechnology, Vol. 43, A. Fiechter, ed. (Springer-Verlag:
Berlin), pp.
11-30.
Conventional methods for the recovery of recombinant protein from the
cytoplasm, as soluble protein or refractile particles, involved disintegration
of the
bacterial cell by mechanical breakage. Mechanical disruption typically
involves the
generation of local cavitations in a liquid suspension, rapid agitation with
rigid beads,
sonication, or grinding of cell suspension (Bacterial Cell Surface Techniques,
Hancock and Poxton (John Wiley & Sons Ltd, 1988), Chapter 3, p. 55).
HEW-lysozyme acts biochemically to hydrolyze the peptidoglycan backbone
of the cell wall. The method was first developed by Zinder and Arndt (1956)
Proc.
Natl. Acad. Sci. USA, 42: 586-590, who treated E. coli with egg albumin (which
contains HEW-lysozyme) to produce rounded cellular spheres later known as
spheroplasts. These structures retained some cell-wall components but had
large
surface areas in which the cytoplasmic membrane was exposed. U.S. Pat. No.
5,169,772 discloses a method for purifying heparinase from bacteria comprising
disrupting the envelope of the bacteria in an osmotically-stabilized medium,
e.g., 20%
sucrose solution using, e.g., EDTA, lysozyme, or an organic compound,
releasing the
non-heparinase-like proteins from the periplasmic space of the disrupted
bacteria by
exposing the bacteria to a low-ionic-strength buffer, and releasing the
heparinase-like
proteins by exposing the low-ionic-strength-washed bacteria to a buffered salt
solution.
Many different modifications of these methods have been used on a wide
range of expression systems with varying degrees of success (Joseph-Liazun et
al.
(1990) Gefae, 86: 291-295; Carter et al. (1992) BiolTeclanology, 10: 163-167).
Efforts
to induce recombinant cell culture to produce lysozyme have been reported. EP
0 155
189 discloses a means for inducing a recombinant cell culture to produce
lysozymes,
which would ordinarily be expected to kill such host cells by means of
destroying or
lysing the cell wall structure.
U.S. Patent No. 4,595,658 discloses a method for facilitating externalization
of
proteins transported to the periplasmic space of E. coli. This method allows
selective
isolation of proteins that locate in the periplasm without the need for
lysozyme
treatment, mechanical grinding, or osmotic shock treatment of cells. U.S.
Patent No.
4,637,980 discloses producing a bacterial product by transforming a
temperature-


CA 02547511 2006-05-29
WO 2005/067478 , PCT/US2004/040117
sensitive lysogen with a DNA molecule that codes, directly or indirectly, for
the
product, culturing the transformant under permissive conditions to express the
gene
product intracellularly, and externalizing the product by raising the
temperature to
induce phage-encoded functions. Asami et al. (1997) J. Ferment. and Bioeng.,
83:
511-516 discloses synchronized disruption of E. coli cells by T4 phage
infection, and
Tanji et al. (1998) .I. Fe~rnent. and Bioeng., 85: 74-78 discloses controlled
expression
of lysis genes encoded in T4 phage for the gentle disruption of E. coli cells.
Upon cell lysis, genomic DNA leaks out of the cytoplasm into the medium and
results in significant increase in fluid viscosity that can impede the
sedimentation of
solids in a centrifugal field. In the absence of shear forces such as those
exerted
during mechanical disruption to break down the DNA polymers, the slower
sedimentation rate of solids through viscous fluid results in poor separation
of solids
and liquid during centrifugation. Other than mechanical shear force, there
exist
nucleolytic enzymes that degrade DNA polymer. In E. coli, the endogenous gene
endA encodes for an endonuclease (molecular weight of the mature protein is
approx.
24.5 kD) that is normally secreted to the periplasm and cleaves DNA into
oligodeoxyribonucleotides in an endonucleolytic manner. It has been suggested
that
endA is relatively weakly expressed by E. coli (Wackernagel et al. (1995) Gene
154:
55-59).
Detection of the expressed protein is achieved by methods known in the art
and include, for example, radioimmunoassays, Western blotting techniques or
immunoprecipitation.
Certain proteins expressed in this invention may form insoluble aggregates
("inclusion bodies"). Several protocols are suitable for purification of
proteins from.
inclusion bodies. For example, purification of inclusion bodies typically
involves the
extraction, separation and/or purification of inclusion bodies by disruption
of the host
cells, e.g., by incubation in a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCI, 5
mM MgCl2, 1 mM DTT, 0.1 mM ATP, and 1 mM PMSF. The cell suspension is
typically lysed using 2-3 passages through a French Press. The cell suspension
can
also be homogenized using a Polytron (Brinkrnan Instruments) or sonicated on
ice.
Alternate methods of lysing bacteria are apparent to those of skill in the art
(see, e.g.,
Sambrook et al., supra; Ausubel et al., supra).
If necessary, the inclusion bodies can be solubilized, and the lysed cell
suspension typically can be centrifuged to remove unwanted insoluble matter.
56


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
Proteins that formed the inclusion bodies may be renatured by dilution or
dialysis
with a compatible buffer. Suitable solvents include, but are not limited to
urea (from
about 4 M to about 8 M), formamide (at least about 80%, volume/volume basis),
and
guanidine hydrochloride (from about 4 M to about 8 M). Although guanidine
hydrochloride and similar agents are denaturants, this denaturation is not
irreversible
and renaturation may occur upon removal (by dialysis, for example) or dilution
of the
denaturant, allowing re-formation of immunologically and/or biologically
active
protein. Other suitable buffers are known to those skilled in the art.
Alternatively, it is possible to purify the recombinant peptides from the host
periplasm. After lysis of the host cell, when the recombinant protein is
exported into
the periplasm of the host cell, the periplasmic fraction of the bacteria can
be isolated
by cold osmotic shock in addition to other methods known to those skilled in
the art.
To isolate recombinant proteins from the periplasm, for example, the bacterial
cells
can be centrifuged to form a pellet. The pellet can be resuspended in a buffer
containing 20% sucrose. To lyse the cells, the bacteria can be centrifuged and
the
pellet can be resuspended in ice-cold 5 mM MgSO4 and kept in an ice bath
for
approximately 10 minutes. The cell suspension can be centrifuged and the
supernatant
decanted and saved. The recombinant proteins present in the supernatant can be
separated from the host proteins by standard separation techniques well known
to
those of skill in the art.
An initial salt fractionation can separate many of the unwanted host cell
proteins (or proteins derived from the cell culture media) from the
recombinant
protein of interest. One such example can be ammonium sulfate. Ammonium
sulfate
precipitates proteins by effectively reducing the amount of water in the
protein
mixture. Proteins then precipitate on the basis of their solubility. The more
hydrophobic a protein is, the more likely it is to precipitate at lower
ammonium
sulfate concentrations. A typical protocol includes adding saturated ammonium
sulfate to a protein solution so that the resultant ammonium sulfate
concentration is
between 20-30%. This concentration will precipitate the most hydrophobic of
proteins. The precipitate is then discarded (unless the protein of interest is
hydrophobic) and ammonium sulfate is added to the supernatant to a
concentration
known to precipitate the protein of interest. The precipitate is then
solubilized in
buffer and the excess salt removed if necessary, either through dialysis or
diafiltration.
Other methods that rely on solubility of proteins, such as cold ethanol
precipitation,
57


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
are well known to those of skill in the art and can be used to fractionate
complex
protein mixtures.
'The molecular weight of a recombinant protein can be used to isolated it from
proteins of greater and lesser size using ultrafiltration through membranes of
different
pore size (for example, Amicon or Millipore membranes). As a first step, the
protein
mixture can be ultrafiltered through a membrane with a pore size that has a
lower
molecular weight cut-off than the molecular weight of the protein of interest.
The
retentate of the ultrafiltration can then be ultra~ltered against a membrane
with a
molecular cut off greater than the molecular weight of the protein of
interest. The
recombinant protein will pass through the membrane into the filtrate. The
filtrate can
then be chromatographed as described below.
Recombinant proteins can also be separated from other proteins on the basis of
its size, net surface charge, hydrophobicity, and affinity for ligands. In
addition,
antibodies raised against proteins can be conjugated to column matrices and
the
proteins immunopurified. All of these methods are well known in the art. It
will be
apparent to one of skill that chromatographic techniques can be performed at
any
scale and using equipment from many different manufacturers (e.g., Pharmacia
Biotech).
Refzaturation aid Refolding
Insoluble protein can be renatured or refolded to generate secondary and
tertiary protein structure conformation. Protein refolding steps can be used,
as
necessary, in completing configuration of the recombinant product. Refolding
and
renaturation can be accomplished using an agent that is known in the art'to
promote
dissociation/association of proteins. For example, the protein can be
incubated with
dithiothreitol followed by incubation with oxidized glutathione disodium salt
followed by incubation with a buffer containing a refolding agent such as
urea.
Recombinant protein can also be renatured, for example, by dialyzing it
against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus
200
mM NaCI. Alternatively, the protein can be refolded while immobilized on a
column,
such as the Ni NTA column by using a linear 6M-1M urea gradient in 500 mM
NaCI,
20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The
renaturation can be performed over a period of 1.5 hours or more. After
renaturation
the proteins can be eluted by the addition of 250 mM immidazole. Imlnidazole
can be
58


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6
buffer
plus 200 mM NaCI. The purified protein can be stored at 4°C or frozen
at -80°C.
Other methods include, for example, those that may be described in MH Lee
et al., Protein Expr. Purif., 25(1): p. 166-73 (2002), W.K. Cho et al., J.
Biotechnology,
77(2-3): p. 169-78 (2000), Ausubel, et al. (1987 and periodic supplements),
Deutscher
(1990) "Guide to Protein Purification," Methods in Enzymology vol. 182, and
other
volumes in this series, Coligan, et al. (1996 and periodic Supplements)
Current
Protocols in Protein Science Wiley/Greene, NY, S. Roe, Protein Purification
Techniques: A Practical Approach (Practical Approach Series), Oxford Press
(2001);
D. Bollag, et al., Protein Methods, Wiley-Lisa, Inc. (1996)
Active Peptide Analysis
Active proteins can have a specific activity of at least 20%, 30%, or 40%, and
preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%,
or 95%
that of the native peptide that the sequence is derived from. Further, the
substrate
specificity (k~at /Kn.,) is optionally substantially similar to the native
peptide.
Typically, k~at /Km will be at least 30%, 40%, or 50%, that of the native
peptide; and
more preferably at least 60%, 70%, 80%, or 90%. Methods of assaying and
quantifying measures of protein and peptide activity and substrate specificity
(k~at
/Km), are well known to those of skill in the art.
The activity of a recombinant peptide produced in accordance with the present
invention by can be measured by any protein specific conventional or standard
in
vitno or in vivo assay known in the art. The activity of the Pseudomonas
produced
recombinant peptide can be compared with the activity of the corresponding
native
protein to determine whether the recombinant protein exhibits substantially
similar or
equivalent activity to the activity generally observed in the native peptide
under the
same or similar physiological conditions.
The activity of the recombinant protein can be compared with a previously
established native peptide standard activity. Alternatively, the activity of
the
recombinant peptide can be determined in a simultaneous, or substantially
simultaneous, comparative assay with the native peptide. For example, an in
vitro
assays can be used to determine any detectable interaction between a
recombinant
peptide and a target, e.g. between an expressed enzyme and substrate, between
expressed hormone and hormone receptor, between expressed antibody and
antigen,
59


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
etc. Such detection can include the measurement of colorimetric changes,
proliferation changes, cell death, cell repelling, changes in radioactivity,
changes in
solubility, changes in molecular weight as measured by gel electrophoresis
and/or gel
exclusion methods, phosphorylation abilities, antibody specificity assays such
as
ELISA assays, etc. In addition, in vivo assays include, but are not limited
to, assays to
detect physiological effects of the Pseudof~aonas produced peptide in
comparison to
physiological effects of the native peptide, e.g. weight gain, change in
electrolyte
balance, change in blood clotting time, changes in clot dissolution and the
induction
of antigenic response. Generally, any ih vitro or ifz vivo assay can be used
to
determine the active nature of the Pseudomofaas produced recombinant peptide
that
allows for a comparative analysis to the native peptide so long as such
activity is
assayable. Alternatively, the peptides produced in the present invention can
be
assayed for the ability to stimulate or inhibit interaction between the
peptide and a
molecule that normally interacts with the peptide, e.g. a substrate or a
component of
the signal pathway that the native protein normally interacts. Such assays can
typically include the steps of combining the protein with a substrate molecule
under
conditions that allow the peptide to interact with the target molecule, and
detect the
biochemical consequence of the interaction with the protein and the target
molecule.
Assays that can be utilized to determine peptide activity are described, for
example, in
Ralph, P. J., et al. (1984) J. Immunol. 132:1858 or Saiki et al. (1981) J.
Immunol.
127:1044, .Steward, W. E. II (1980) The Interferon Systems. Springer-Verlag,
Vienna
and New York, Broxmeyer, H. E., et al. (1982) Blood 60:595, "Molecular
Cloning: A
Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J.,
E. F.
Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to
Molecular
Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimrnel eds.,
1987,
AK Patra et al., Protein Expr Purif, 18(2): p/ 182-92 (2000), Kodama et al.,
J.
Biochem. 99: 1465-1472 (1986); Stewart et al., Proc. Nat'1 Acad. Sci. USA 90:
5209-
5213 (1993); (Lombillo et al., J. Cell Biol. 128:107-115 (1995); (Vale et al.,
Cell
42:39-50 (1985).
EXAMPLES
In these examples, the cowpea chlorotic mottle virus (CCMV) has been used
as a peptide carrier and Pseudomohas fluorescefas has been used as the
expression
host. CCMV is a member of the bromovirus group of the Bromoviridae.


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
Bromoviruses are 25-28 nm diameter icosahedral viruses with a four-component,
positive sense, single-stranded RNA genome. RNA1 and RNA2 code for replicase
enzymes. RNA3 codes for a protein involved in viral movement within plant
hosts.
RNA4 (a subgenomic RNA derived from RNA 3), i.e. sgRNA4, codes for the 20 l~Da
capsid (CP), SEQ ID NO:1.
Wild type CCMV c~sid encoded by sgRRNA4 (SEQ ID NO:11
Met Ser Thr Val Gly Thr Gly Lys Leu Thr Arg Ala Gln Arg Arg Ala Ala Ala
Arg Lys Asn Lys Arg Asn Thr Arg Val Val Gln Pro Val Ile Val Glu Pro Ile Ala
Ser Gly Gln Gly Lys Ala Ile Lys Ala Trp Thr Gly Tyr Ser Val Ser Lys Trp Thr
Ala Ser Cys Ala Ala Ala Glu Ala Lys Val Thr Ser Ala Ile Thr Ile Ser Leu Pro
Asn Glu Leu Ser Ser Glu Arg Asn Lys Gln Leu Lys Val Gly Arg Val Leu Leu
Trp Leu Gly Leu Leu Pro Ser Val Ser Gly Thr Val Lys Ser Cys Val Thr Glu Thr
Gln Thr Thr Ala Ala Ala Ser Phe Gln Val Ala Leu Ala Val Ala Asp Asn Ser Lys
Asp Val Val Ala Ala Met Tyr Pro Glu Ala Phe Lys Gly Ile Thr Leu Glu Gln Leu
Thr Ala Asp Leu Thr Ile Tyr Leu Tyr Ser Ser Ala Ala Leu Thr Glu Gly Asp Val
Ile Val His Leu Glu Val Glu His Val Arg Pro Thr Phe Asp Asp Ser Phe Thr Pro
Val Tyr
Each CCMV particle contains up to about 180 copies of the CCMV CP. An
exemplary DNA sequence encoding the CCMV CP is shown in SEQ ~ NO: 21.
Exemplary DNA seguence encodingthe CCMV CP (SEQ ID N0:21)
atg tct aca gtc gga aca ggg aag tta act cgt gca caa cga agg get gcg gcc cgt
aag aac
aag cgg aac act cgt gtg gtc caa cct gtt att gta gaa ccc atc get tca ggc caa
ggc aag
get att aaa gca tgg acc ggt tac agc gta tcg aag tgg acc gcc tct tgc gcg gcc
gcc gaa
get aaa gta acc tcg get ata act atc tct ctc cct aat gag cta tcg tcc gaa agg
aac aag
cag ctc aag gta ggt aga gtt tta tta tgg ctt ggg ttg ctt ccc agt gtt agt ggc
aca gtg aaa
tcc tgt gtt aca gag acg cag act act get get gcc tcc ttt cag gtg gca tta get
gtg gcc gac
aac tcg aaa gat gtt gtc get get atg tac ccc gag gcg ttt aag ggt ata acc ctt
gaa caa ctc
acc gcg gat tta acg atc tac ttg tac agc agt gcg get ctc act gag ggc gac gtc
atc gtg
cat ttg gag gtt gag cat gtc aga cct acg ttt gac gac tct ttc act ccg gtg tat
tag
The crystal structure of CCMV has been solved. This structure provides a
clearer picture of the capsid interactions that appear to be critical to
particle stability
and dynamics and has been helpful in guiding rational design of insertion
sites.
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Previous studies have demonstrated that CCMV capsids can be genetically
modified
to carry heterologous peptides without interfering with their ability to form
particles.
A number of suitable insertion sites have been identified.
The general strategy followed for production of capsid-peptide fusion VLPs in
P. fluorescens is diagrammed in Figure 2. A total of up to about 180 copies of
a
heterologous peptide unit (whether individual peptide or concatemer) can be
inserted
into the CCMV particle if a single insertion site in the CCMV CP is used.
Insertion
sites identified within CCMV CP to date can accommodate peptides of various
lengths. In addition, multimeric forms of the peptides can be inserted into
insertion
sites. Furthermore, multiple insertion sites can be used at the same time to
express the
same or different peptides in/on the same particle. The peptide inserts can be
about
200 amino acid residues or less in length, more preferably up to or about 180,
even
more preferably up to or about 150, still more preferably up to or about 120,
yet more
preferably up to or about 100 amino acid residues in length. In a preferred
embodiment, the peptide inserts will be about 5 or more amino acid residues in
length.
In a preferred embodiment, the peptide inserts will be about 5 to about 120,
more
preferably about 5 to about 100 amino acid residues in length.
Materials and Methods
Unless otherwise noted, standard techniques, vectors, control sequence
elements, and other expression system elements known in the field of molecular
biology are used for nucleic acid manipulation, transformation, and
expression. Such
standard techniques, vectors, and elements can be found, for example, in:
Ausubel et
al. (eds.), Cuy~rent Protocols irZ Molecular Biology (1995) (John Wiley &
Sons);
Sambrook, Fritsch, & Maniatis (eds.), Molecular Cloning (1989) (Cold Spring
Harbor
Laboratory Press, NY); Berger & Kimmel, Metlaods in Enzynaology 152: Guide to
Molecular Cloning Techniques (1987) (Academic Press); and Bukhari et al.
(eds.),
DNA Insertion Elernents, Plasmids and Episornes (1977) (Cold Spring Harbor
Laboratory Press, NY).
Plasmid Map Constructions
All plasmid maps were constructed using VECTORNTI (InforMax Inc.,
Frederick, MD, USA).
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DNA Extractions
All plasmid DNA extractions from E. coli were performed using the mini,
midi, and maxi kits from Qiagen (Germany) according to the manufacturer
instructions.
Experimental Strate~y
The following procedures were followed. P. fluorescens host cells were
transformed with expression plasmids encoding chimeric viral capsid-target
peptide
insert fusions. Transformed cells were grown to the desired density and
induced to
express the chimeric viral capsid-peptide fusions. Cells were then lysed and
their
contents analyzed.
Construction of Modified CCMV-CP DNA to Add an Engineered Insertion Site
A DNA molecule containing the CCMV CP coding sequence was modified by
inserting, in reading frame, a BarnHI restriction enzyme recognition and
cleavage site
(gggatcctn), which introduced a tripeptide (Gly-Ile-Leu), into the native CCMV-
CP
amino acid sequence, between Asn129 and Ser130. Thus, the native CCMV-CP
amino acid sequence (SEQ ID NO:1) was modified to form CCMV129-CP (SEQ ID
N0:2).
CCMV CP with added BamHI site inserted at codon 129 (CCMV129-CP) (SEQ ID
N0:2
Met Ser Thr Val Gly Thr Gly Lys Leu Thr Arg Ala Gln Arg Arg Ala Ala Ala
Arg Lys Asn Lys Arg Asn Thr Arg Val Val Gln Pro Val Ile Val Glu Pro Ile Ala
Ser Gly Gln Gly Lys Ala Ile Lys Ala Trp Thr Gly Tyr Ser Val Ser Lys Trp Thr
Ala Ser Cys Ala Ala Ala Glu Ala Lys Val Thr Ser Ala Ile Thr Ile Ser Leu Pro
Asn Glu Leu Ser Ser Glu Arg Asn Lys Gln Leu Lys Val Gly Arg Val Leu Leu
Trp Leu Gly Leu Leu Pro S er V al S er Gly Thr V al Lys S er Cys V al Thr Glu
Thr
Gln Thr Thr Ala Ala Ala Ser Phe Gln Val Ala Leu Ala Val Ala Asp Asn Gly Ile
Leu Ser Lys Asp Val Val Ala Ala Met Tyr Pro Glu Ala Phe Lys Gly Ile Thr Leu
Glu Gln Leu Thr Ala Asp Leu Thr Ile Tyr Leu Tyr Ser Ser Ala Ala Leu Thr Glu
Gly Asp Val Ile Val His Leu Glu Val Glu His Val Arg Pro Thr Phe Asp Asp Ser
Phe Thr Pro Val Tyr
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Primer CCMV-For (nucleic acid sequence: 5'-gactagtagg aggaaagaga
tgtctacagt cgg -3'(SEQ ID NO:3)) was designed to add an ACTAGT ,SpeI
restriction
site and to add the consensus Shine-Dalgarno sequence to the CCMV-CP coding
sequence. Primer CCMV-Rev (nucleic acid sequence: 5'-ccgctcgagt cattactaat
acaccgg-3' (SEQ ID NO:4)) was designed to add a CTCGAG ~'hoI restriction site
and
to introduce two stop codons to the CCMV-CP coding sequence. These two primers
were used in a first PCR reaction with the DNA coding sequence of CCMV-CP.
Construction of CCMV63 -CP DNA to Add an Engineered Insertion Site:
Restriction sites AscI and NotI were engineered onto CCMV-CP (SEQ ID
NO:1) to serve as an insertion site. Recognition and cleavage sites for AscI
(ggcgcgcc), NotI (gcggccgc), and additional nucleotides introduced a
heptapeptide
(Glu-Ala-Trp-Arg-Ala-Ala-Ala) onto CCMV-CP between residue Ala 60 and Ala 61.
Hence, CCMV-CP was modified to form CCMV63-CP. In addition, residue Arg 26
was mutated to Cys 26 to add stability to assembled VLPs.
The plasmid map of pCCMV63-CP is shown in Figure 4.
Seduence of CCMV63-CP ORF (SEQ ID NO: 22):
atgtctacagtcggaacagggaagttaactcgtgcacaacgaagggctgcggcccgtaagaacaagcggaacacttgt
gtggtccaacctgttattgtagaacccatcgcttcaggccaaggcaaggctattaaagcatggaccggttacagcgtat
c
gaagtggaccgcctcttgtgcggctgccgaagcttggcgcgccgcggccgctaaagtaacctcggctataactatctct

ctccctaatgagctatcgtccgaaaggaacaagcagctcaaggtaggtagagttttattatggcttgggttgcttccca
gtg
ttagtggcacagtgaaatcctgtgttacagagacgcagactactgctgctgcctcctttcaggtggcattagctgtggc
cg
acaactcgaaagatgttgtcgctgctatgtaccccgaggcgtttaagggtataacccttgaacaactcaccgcggattt
aa
cgatctacttgtacagcagtgcggctctcactgagggcgacgtcatcgtgcatttggaggttgagcatgtcagacctac
gt
ttgacgactctttcactccggtgtattagtaatga
Construction of double insertion R26C-CCMV63/129-CP:
Restriction sites AscI and NotI were engineered onto CCMV 129-CP (SEQ ID N0:2)
to serve as the second insertion site. Recognition and cleavage sites for AscI
(ggcgcgcc), NotI (gcggccgc), and additional nucleotides introduced a
heptapeptide
(Glu-Ala-Trp-Arg-Ala-Ala-Ala) onto CCMV129-CP between residue Ala 60 and Ala
61. Hence, CCMV129-CP was modified to form CCMV63/129-CP. In addition,
residue Arg 26 was mutated to Cys 26 to add stability to assembled VLPs to
create
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R26C-CCMV63/129-CP. The plasmid map of pR26C-CCMV63/129-CP is shown in
Figure 5.
Sequence of R26C-CCMV63/129-CP ORF (SEQ ID NO: 23~
atgtctacagtcggaacagggaagttaactcgtgcacaacgaagggctgcggcccgtaagaacaagcggaacactt
gtgtggtccaacctgttattgtagaacccatcgcttcaggccaaggcaaggctattaaagcatggaccggttacagcgt

atcgaagtggaccgcctcttgtgcggctgccgaagcttggcgcgccgcggccgctaaagtaacctcggctataacta
tctctctccctaatgagctatcgtccgaaaggaacaagcagctcaaggtaggtagagttttattatggcttgggttgct
tc
ccagtgttagtggcacagtgaaatcctgtgttacagagacgcagactactgctgctgcctcctttcaggtggcattagc
t
gtggccgacaacgggatcctgtcgaaagatgttgtcgctgctatgtaccccgaggcgtttaagggtataacccttgaac

aactcaccgcggatttaacgatctacttgtacagcagtgcggctctcactgagggcgacgtcatcgtgcatttggaggt

tgagcatgtcagacctacgtttgacgactctttcactccggtgtattagtaatga
Example 1: Production of Peptide PDl in CCMV VLPs in Pseudo~notzas
l .A. Construction of the Chimeric CCMV-PD 1 Gene
A 20 amino acid antigenic peptide was selected for expression as an insert in
the CCMV viral capsid. The antigenic peptide was unrelated to CCMV and to
Pseudomonas fluor~escefzs. An oligonucleotide encoding the peptide was
amplified out
of plasmid pCP7Parvol DNA using primers Parvo-BamHI-F (nucleic acid sequence:
5'-cgggatcctg gacccggatg-3' (SEQ ID N0:16)) and Parvo-BamHI-R (nucleic acid
sequence: 5'-cgggatcccc gggtctcttt c-3' (SEQ ID N0:17)). (These primers were
obtained from Integrated DNA Technologies, Inc., Coralville, IA, USA,
hereinafter
"IdtDNA.") These primers amplified out a Canine parvovirus peptide coding
sequence
while adding BamHI restriction sites thereto at both ends for insertion into
the
CCMV129 coding sequence, at the BanzHI restriction site thereof.
The PCR reactions were performed using a PTC225 thermocycler (MJ
Research, South San Francisco, CA, USA) according to the following protocol:
Table 4. PCR PROTOCOL
Reaction Mix (100uL total volume) Thermocycling Steps
10 ~,L 10X PT HIFI buffer * Step 1 Cycle min. 94C
1~


~,L SOmM MgS04 * 30 sec.94C


,uL lOmM dNTPs * Step 35 Cycles30 sec.55C
2


0.25 ng ach Primer 1 min. 68C




CA 02547511 2006-05-29
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1-5 ng emplate DNA Step 1 Cycle 10 70C
3 min.


1 ~,L T HIFI Taq DNA PolymeraseStep 1 Cycle Maintain4C
* 4


emainder distilled De-ionized H20
(ddH20)


* (from Invitrogen Corp, Carlsbad, C;A, USA, hereinafter "lnmtrogen-
The DNA sequence was inserted into the CCMV 129 shuttle plasmid, a
plasmid that had been constructed from plasmid pESC (obtained from Stratagene
Corp., LaJolla, CA, USA) by inserting nucleic acid containing the CCMV 129 CP
DNA sequence therein, by use of SpeI and XhoI restriction enzymes. The PDl
peptide-encoding nucleic acid was inserted at the BamHI restriction site
within the
CCMV129 CDS, producing the CCMV129-PD1 shuttle plasmid.
PDl CDS was also inserted into the CCMV129 CDS. As a result, the inserted
PD1 coding sequence is: 5'-tgg gcc tgc cgc ggc acg gcc ggc tgg ccg ccg tcc ggc
tgc
acg gcg ccg tcc ggg tcg-3' (SEQ ID N0:18), encoding a PDl peptide whose amino
acid sequence is: Trp Ala Cys Arg Gly Thr Ala Gly Trp Pro Pro Ser Gly Cys Thr
Ala
Pro Ser Gly Ser (SEQ ID N0:7). The PD1-coding nucleotide sequence is unrelated
to
Canine parvovirus.
1 B Construction of a CCMV-PD1 Expression Plasmid
The CCMVl29-PDl shuttle plasmid was digested with SpeI and XhoI
restriction enzymes. The fragment containing the chimeric CCMV129-PDl DNA
sequence was isolated by gel purification. It was then inserted into the
pMYC1803
expression plasmid, in place of the buibui toxin gene, in operable attachment
to a tac
promoter, at the expression plasmid's SpeI and XhoI restriction sites. See
Figure 1.
The resulting expression plasmid was screened by restriction enzyme digestion
with
SpeI and XhoI to verify the presence of the insert.
1.C. Plasmid Transformation into Pseudomonas Host Cells
The CCMV129-PD1 expression plasmid was transformed into Pseudomoraas
fluorescens MB214 host cells according to the following protocol. Host cells
were
thawed gradually in vials maintained on ice. For each transformation, 1 ~,L
purified
expression plasmid DNA was added to the host cells and the resulting mixture
was
swirled gently with a pipette tip to mix, and then incubated on ice for 30
min. The
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mixture was transferred to electroporation disposable cuvettes (BioRad Gene
Pulser
Cuvette, 0.2 cm electrode gap, cat no. 165-2086). The cuvettes were placed
into a
Biorad Gene Pulser pre-set at 200 Ohms, 25~.farads, 2.25kV. Cells were pulse
cells
briefly (about 1-2 sec). Cold LB medium was then immediately added and the
S resulting suspension was incubated at 30°C for 2 hours. Cells were
then plated on LB
tetl5 (tetracycline-supplemented LB medium) agar and grown at 30°C
overnight.
1 D Shake-Flask Expression of CCMV-PDl Construct
One colony was picked from each plate and the picked sample was inoculated
into SOmL LB seed culture in a baffled shake flask. Liquid suspension cultures
were
grown overnight at 30°C with 250rpm shaking. lOmL of each resulting
seed culture
was then used to inoculate 200mL of shake-flask medium (i.e. yeast extracts
and salt
with trace elements, sodium citrate, and glycerol, pH 6.8) in a 1 liter
baffled shake
flask. Tetracycline was added for selection. Inoculated cultures were grown
overnight
at 30°C with 250rpm shaking and induced with IPTG for expression of the
CCMV 129-PD 1 chimeric capsids.
1 E S ~aration of Cell Culture Lysate into Soluble and Insoluble Fractions
1 mL aliquots from each shake-flask culture were then centrifuged to pellet
the
cells. Cell pellets were resuspended in 0.75mL cold 50mM Tris-HCI, pH 8.2,
containing 2mM EDTA. 0.1% volume of 10% TritonX-100 detergent was then added,
followed by an addition of lysozyme to 0.2mg/mL final concentration. Cells
were
then incubated on ice for 2 hours, at which time a clear and viscous cell
lysate should
be apparent.
To the lysates, 1/200 volume 1M MgCl2 was added, followed by an addition
of 1/200 volume 2mg/mL DNAseI, and then incubation on ice for 1 hour, by which
time the lysate should have become a much less viscous liquid. Treated lysates
were
then spun for 30 min at 4°C at maximum speed in a tabletop centrifuge
and the
supernatants were decanted into clean tubes. The decanted supernatants are the
"soluble" protein fractions. The remaining pellets were then resuspended in
0.75 mL
TE buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA). The resuspended pellets are the
"insoluble" fractions.
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1 F SDS-PAGE and Western Blot Analysis of Soluble and Insoluble Protein
Fractions
These "soluble" and "insoluble" fractions were then electrophoresed on
NuPAGE 4-12% Bis-Tris gels (from Invitrogen, Cat. NP0323), having l.Omm x 15
wells, according to manufacturer's specification. Gels were stained with
SimplyBlue
Safe Stain, (from Invitrogen, Cat. LC6060) and destained overnight with water.
Western blot detection employed CCMV IgG (Accession No. AS0011 from DSMZ,
Germany) and the WESTERN BREEZE kit (from Invitrogen, Cat. WB7105),
following manufacturer's protocols. Results were positive for production of
CCMV
and specifically for production of chimeric CCMV CP (see Figure 6 and 7).
1 G PEG Preci~tation of Chimeric VLPs
Chimeric, i.e. recombinant, VLPs were precipitated by lysis of separate shake
flask culture samples, followed by PEG(polyethylene glycol)-treatment of the
resulting cell lysates, according to the following protocol. SmL aliquots of
each
shake-flask culture were centrifuged to pellet the cells. Pelleted cells were
resuspended in O.1M phosphate buffer (preferably a combination of monobasic
and
dibasic potassium phosphate), pH 7.0, at a 2 volume buffer to 1 volume pellet
ratio.
Cells were then sonicated for 10 sec, 4 times, with 2 minutes resting on ice
in between.
During this sonication procedure, the cell lysate should clear somewhat.
Following
sonication, lysozyme was added to final concentration of O.Smg/mL. Lysozyme
digestion was allowed to proceed for 30 min at room temperature.
The resulting treated lysates were then centrifuged for 5 min at 15000xG at
4°C. The resulting supernatants were removed and their volumes
measured. To each
supernatant, PEG6000 was added to a final concentration of 4%; followed by
NaCl
addition to a final concentration of 0.2M, and incubation on ice for 1 hr or
overnight
at 4°C. Then, these were centrifuged at 20000xG for 15 min at
4°C. Precipitated
pellets were then resuspended in 1/10 initial supernatant volume of phosphate
buffer
and stored at 4°C.
1.H. Sucrose Gradient Centrifugation
Sucrose solutions were made with sucrose (Sigma, Cat. S-5390) in phosphate
buffer. Sucrose gradients were poured manually 10%, 20%, 30%, and 40% from top
to bottom. The resuspended precipitated pellet samples were then spun in a
Beckman-
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Coulter SW41-Ti rotor in a Beckman-Coulter Optima XL 100K Ultracentrifuge for
1
hour with no braking. Each 1mL fraction of the sucrose gradient was eluted
separately
and further spun down to obtain VLP pellets. VLP pellets were resuspended in
phosphate buffer, electrophoresed on SDS-PAGE gels, and Western blotted using
CCMV IgG as per the above protocol. Western blot was positive for VLP
formation
(Figure 8). A portion of each resulting VLP preparation was used for electron
microscopy.
1.I. Electron Microscopy Analysis
VLP samples were spotted on either collodion/carbon- or formvar/carbon
coated grids. Samples were stained with 2% phosphotungstic acid (PTA) and were
imaged on a Philips CM-12 TEM transmission electron microscope (Serial #D769),
operated at an accelerating voltage of 120 kV. Images were recorded digitally
with a
MultiScan CCD camera (from Gatan, Inc., Pleasanton, CA, USA; Model 749, Serial
#
971119010). Formation of VLPs was verified (Figure 9).
Example 2: Production of DZA21 AMP Trimers in CCMV VLPs in Pseudomonas
and Recovery of AMPs Therefrom
2 A. Synthesis of D2A21 insert:
A nucleotide sequence coding an anti-microbial peptide ("AMP") trimer
("D2A21 trimer," i.e. containing three D2A21 monomeric AMPS) was amplified out
of plasmid pET-(D2A21)3 using primers D2A21-BamHI-F (nucleic acid sequence:
5'-cgggatcctg ggacagcaaa tgggtcgcga tccg-3' (SEQ ID NO:S)) and D2A21-BamHI-R
(nucleic acid sequence: 5'-cgggatcccg tcgacggagc tcgaattcgg atcacc-3' (SEQ ID
N0:6)). PCR reactions were performed according to the same protocols as
described
in Example 1.A., above.
The resulting amplified insert contained a BarnHI restriction site added at
each
end, for use in inserting the D2A21 trimer CDS into the CCMV 129 CDS at the
engineered BamHI site. The nucleotide sequence encoding, and the amino acid
sequence of, the D2A21 trimer are shown in SEQ ID NOs:l9 and 20, respectively.
Nucleotide sequence encoding the D2A21 trimer (SEQ ID N0:191:
5'-ttc gcg aag aag ttt gcg aaa aag ttc aag aaa ttt gcc aag aag ttt gcc aag ttc
gca ttc
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gcg ttc ggc gat ccg ttc gcg aag aag ttt gcg aaa aag ttc aag aaa ttt gcc aag
aag ttt
gcc aag ttc gca ttc gcg ttc ggc gat ccg ttc gcg aag aag ttt gcg aaa aag ttc
aag aaa
ttt gcc aag aag ttt gcc aag ttc gca ttc gcg ttc ggt -3'
Amino acid sequence of the D2A21 trimer (SEQ ID N0:20):
Phe Ala Lys Lys Phe Ala Lys Lys Phe Lys Lys Phe Ala Lys Lys Phe Ala Lys
Phe Ala Phe Ala Phe Gly Asp Pro Phe Ala Lys Lys Phe Ala Lys Lys Phe Lys
Lys Phe Ala Lys Lys Phe Ala Lys Phe Ala Phe Ala Phe Gly Asp Pro Phe Ala
Lys Lys Phe Ala Lys Lys Phe Lys Lys Phe Ala Lys Lys Phe Ala Lys Phe Ala
Phe Ala Phe Gly
The trimer CDS contained the three AMP monomer CDSs separated by di-
peptide Asp-Pro acid-labile cleavage site CDSs, as shown in Figure 3. The
entire
trimer CDS was also bordered at each terminus by an dipeptide Asp-Pro acid-
labile
cleavage site CDS. The amplified insert was digested with BaynHI restriction
enzyme
to create adhesive ends for cloning into the pESC-CCMV129BamHI shuttle plasmid
at the BanaHI site within the CCMV129 CDS. The resulting shuttle plasmid was
digested with SpeI and XhoI restriction enzymes. The desired chimeric RBS/CDS
fragment was isolated by gel purification.
2 B E~ression Plasmid Construction
The resulting chimeric CCMV129-(D2A21)3 polynucleotide was then inserted
into the pMYC1803 expression plasmid in place of the buibui coding sequence,
in
operable attachment to the tac promoter. The resulting expression plasmid was
screened by restriction digest with SpeI and XhoI for presence of the insert.
The same
protocols as described above for Example 1B were utilized.
2 C Transformation and Expression
The resulting expression plasmid was transformed into P. fluo~escens MB214,
using the protocol described above for Example 1.C. Plate-colonized
transformants
were picked and transferred to shake flasks for expression, following the same
protocol as described for Example 1.D., above.
2 D Protein and VLP Recovery and Analysis


CA 02547511 2006-05-29
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Shake-flask-cultured cells were lysed and fractionated, following the
procedures of Example 1.E. The resulting fractions were analyzed by SDS-PAGE
and Western blotting as described for Example 1.F. Chimeric VLPs were
recovered
by PEG precipitation and sucrose gradient centrifugation, and analyzed by
electron
microscopy, as described above for Examples 1.G. through 1.I. Chimeric CCMV
VLP
assembly was verified. Results were positive for the proaucuon of ~,m~ v .
,~~a-
PAGE for chimeric CP expression showed a 96 amino acids insert (Figure 10),
which
was confirmed by western blot after fractionation on sucrose gradient to show
VLP
formation (Figure 11) and by electron micrograph to verify VLP formation
(Figure
12).
2 E Analysis of D2A21 Anti-Microbial Peptide Production
Soluble and insoluble protein fractions were further treated to characterize
D2A21 peptides produced in the chimeric VLPs, as follows.
2.E.1. Acid cleavage of D2A21
The insoluble fraction was dissolved in 15% v/v aqueous acetonitrile and
approximately 40- 50% v/v aqueous formic acid; the soluble fraction was
resuspended
in approximately 45-50% formic acid. The samples were then incubated at
60°C for
24 hours to permit acid cleavage to proceed. The reactions were stopped by
freezing
to -20°C, at which temperature the treated samples were stored until
HPLC analysis.
2.E.2. D2A21 Analysis by Hl'LC
Soluble fractions were filtered through a 0.22~,m membrane; insoluble
fractions were centrifuged to precipitate cellular debris and then filtered
through a
0.22~,m membrane. SO~,L of each sample was added to 950~uL of 25% aqueous
acetonitrile. A volume of 250,uL of each sample, containing a D2A21' internal
control peptide (10~,g control peptide total), was injected onto a VYDAC 250mm
reverse-phase C18 column, 6.4mm internal diameter (available from Grace Vydac,
Hesperia, CA, USA), installed in a Beckman high performance liquid
chromatography (HPLC) system. Elution was performed using an aqueous gradient
of
25% acetonitrile/0.1% trifluoroacetic acid (TFA) to 75% acetonitrile/0.01% TFA
over
30 minutes. Eluates were drip-collected into chromatography fractions. The
appropriate peptide peak was observed only in the sample derived from VLPs
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CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
containing the engineered peptide but not in the sample derived from non-
engineered
VLPs (Figure 13).
2.E.3. Mass Spectrometry Analysis of D2A21 Peptides
Mass spectrometry analysis of peptide controls and of chromatography
fractions were performed using a Micromass M a~LDI linear matrix-assisted
laser
desorption ionizationtime-of flight (MALDI-TOF) mass spectrometry system (from
Micromass UK Ltd., Manchester, UK). Before MS analysis, the HPLC fractions
were
concentrated by centrifugal evaporation, using a Speed Vac system (available
from
Thermo Savant, Milford, MA, USA; model 250DDA). The results demonstrated the
accurate production of D2A21 AMPs and that the peptide that is released is the
D2A21 peptide (Figure 14). These results demonstrated that the use of a VLP
fusion
for peptide expression in Pseudomonads was effective avoided otherwise normal
host-
cell toxicity. Production of (A) chimeric VLPs has been demonstrated and
production
of (B) peptide multimers has been demonstrated in the VLP format (up to 96
amino
acids total). Thus, this Example has:
(1) tested the ability of P. fluorescens to support CCMV CP expression and
particle assembly,
(2) purified chimeric VLPs by a simple method (PEG precipitation),
(3) cleaved off the peptides of interest by previously tested methods (acid
hydrolysis) and
(4) verified the peptide identity and integrity.
Example 3: Production of Anthrax Antigens in CCMV VLPs in Pseudomouas
3.A. Synthesis of PA Peptide Inserts
Four different Bacillus anthracis protective antigen ("PA") peptides (PA1-
PA4) were independently expressed in CCMV VLPs. Nucleic acids encoding PAl-
PA4 were synthesized by SOE (splicing-by-overlap-extension) of synthetic
oligonucleotides. The resulting nucleic acids contained BamHI recognition site
termini. The nucleotide sequences encoding, and the amino acid sequences of,
these
PA peptides were respectively as follows: 1) for PAl, SEQ ID NOs:8 and 9; 2)
for
PA2, SEQ 117 NOs:lO and 11; 3) for PA3, SEQ ID NOs:l2 and 13; and 4) for PA4,
SEQ ID NOs:l4 and 15. The resulting nucleic acids were digested with BarnHI to
create adhesive ends for cloning into shuttle vector. Each of the resulting PA
inserts
72


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
was cloned in the pESC-CCMV129BamHI shuttle plasmid at the BarnHI site of the
CCMV129 CDS. Each resulting shuttle plasmid was digested with SpeI and XhoI
restriction enzymes. Each of the desired chimeric CCMV129-PA-encoding
fragments
was isolated by gel purification.
PAl
Nucleic Acid 5'-agt aat tct cgt aag aaa cgt tct acc tct
get ggc cct acc gtg cct


Sequence (SEQ gat cgt gat aat gat ggc att cct gat-3'
ID


NO:B)


Amino Acid SequenceSer Asn Ser Arg Lys Lys Arg Ser Thr Ser
Ala Gly Pro Thr


(SEQ ID N0:9) Val Pro Asp Arg Asp Asn Asp Gly Ile Pro
Asp


PA2
Nucleic Acid 5'-agt cct gaa get cgt cat cct ctc gtg get
gcg tat cct att gtg cat


Sequence (SEQ gtt gat atg gaa aat att atc ctc tct-3'
ID


NO:10)


Amino Acid SequenceSer Pro Glu Ala Arg His Pro Leu Val Ala
Ala Tyr Pro Ile


(SEQ ID NO:11) Val His Val Asp Met Glu Asn Ile Ile Leu
Ser


PA3
Nucleic Acid 5'-cgt att att ttc aat ggc aaa gat ctc aat
ctc gtg gaa cgt cgt att


Sequence (SEQ get get gtg aat cct tct gat cct ctc -3'
ID


NO: 12)


Amino Acid SequenceArg Ile Ile Phe Asn Gly Lys Asp Leu Asn
Leu Val Glu Arg


(SEQ ID NO: 13) Arg Ile Ala Ala Val Asn Pro Ser Asp Pro
Leu


PA4
Nucleic Acid 5'-cgt caa gat ggc aaa acc ttc att gat ttc
aaa aag tat aat gat aaa


Sequence (SEQ ctc cct ctc tat att tct aat cct aat-3'
ID


N0:14)


Amino Acid SequenceArg Gln Asp Gly Lys Thr Phe Ile Asp Phe
Lys Lys Tyr Asn


(SEQ ID NO: 15) Asp Lys Leu Pro Leu Tyr Ile Ser Asn Pro
Asn


3.B. Expression Plasmid Construction
73


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
The resulting chimeric CCMV 129-PA polynucleotides were each then inserted
into the pMYC1803 expression plasmid in place of the buibui coding sequence,
in
operable attachment to the tac promoter. The resulting expression plasmid was
screened by restriction digest with SpeI and XhoI for presence of the insert.
The same
protocols as described above for Example 1B were utilized.
3.C. Transformation and Expression
The resulting expression plasmid was transformed into P. fluorescefzs MB214,
using the protocol described above for Example 1.C. Plate-colonized
transformants
were picked and transferred to shake flasks for expression, following the same
protocol as described for Example 1.D., above.
3 D Protein and VLP Recovery and Analysis
Shake-flask-cultured cells were lysed and fractionated, following the
procedures of Example 1.E. The resulting fractions were analyzed by SDS-PAGE
and
Western blotting as described for Example 1.F. Results were positive for the
production of CCMV.
Results were positive for chimeric CCMV CP production (see Figure 15).
VLPs were recovered by PEG precipitation and sucrose gradient fractionation as
described in the example 1.G. and 1. H. Western blot of sucrose gradient
fraction was
performed as described in 1.H. The results were positive for VLP formation
(Figure
16).
Example 4: Production of PBF20 AMP Monomers by Single and Double
Insertion into CCMV VLPs in Psedomosaas
The procedures set forth in Examples 1, 2, and 3 were followed. Nucleic acid
encoding PBF20 monomeric peptides (encoding AMPS comprising the amino acid
sequence 3-22 of amino acid sequence Asp Pro Lys Phe Ala Lys Lys Phe Ala Lys
Lys Phe Ala Lys Lys Phe Ala Lys Lys Phe Ala Lys Asp Pro (SEQ ID N0:24)) and
the acid cleavage sites comprising the amino acid sequence 1-2 and 23-24 of
SEQ ID
N0:24 was inserted individually into CCMV63 -CP at AscIlNotI site and
CCMV 129 -CP at BamHI site. The peptide was also inserted into R26C-
CCMV63/129-CP both at the AscIlNotI site and BarnHI site at the same time.
74


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
The resulting chimeric polynucleotides were each then inserted into the pMYC
103 expression plasmid in place of the buibui coding sequence, in operable
attachment to the tac promoter. The resulting expression plasmid was screened
by
restriction digest with SpeI and XlaoI for presence of the insert and
transformed into P.
fluorescefis MB214, using the protocol described above for Example 1.C. Plate-
colonized transformants were picked and transferred to shake flasks for
expression,
following the same protocol as described for Example 1.D., above.
Shake-flask-cultured cells were lysed and fractionated, following the
procedures of Example 1.E. The resulting fractions were analyzed by SDS-PAGE
and Western blotting as described for Example 1.F. Results were positive for
production of VLPs.
SDS-PAGE showing expression of chimeric CCMV63 -CP engineered to
express a 20 amino acid antimicrobial peptide PBF20 separated by acid
hydrolysis
sites in Pseudomohas fluorescens is shown in Figure 17. The chimeric CCMV63 -
CP-PBF20 has slower mobility compared to the non-engineered wild type (wt)
CCMV CP. Electron microscopy (EM) image of chimeric CCMV VLPs derived
from CCMV63-CP and displaying a 20 amino acid antimicrobial peptide PBF20
separated by acid hydrolysis sites is shown in Figure 1 ~.
SDS-PAGE showing expression of chimeric CCMV129 -CP engineered to
express a 20 amino acid antimicrobial peptide PBF20 separated by acid
hydrolysis
sites in Pseudomonas fluof°escefZS is shown in Figure 19. The chimeric
CCMV129
CP-PBF20 has slower mobility compared to the non-engineered wild type (wt)
CCMV CP. Electron microscopy (EM) image of chimeric CCMV VLPs derived
from CCMV129-CP and displaying a 20 amino acid antimicrobial peptide PBF20
separated by acid hydrolysis sites is shown in Figure 20.
SDS-PAGE showing expression of chimeric CCMV63/129 CP engineered to
express a 20 amino acid antimicrobial peptide PBF20 separated by acid
hydrolysis
sites in two different insertion sites in the CP in Pseudomoraas fluor-escef2s
is shown in
Figure 21. Chimeric CP containing a double insert (CP + 2x20 AA) has slower
mobility on the SDS-PAGE gel compared to the capsid engineered to express a
single
insert (CP + 1x20 AA) of the same peptide. Electron microscopy (EM) image of
chimeric CCMV VLPs derived from CCMV63/129-CP displaying a 20 amino acid
antimicrobial peptide PBF20 separated by acid hydrolysis sites in two
insertion sites


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
per capsid is shown in Figure 22: Each VLP was found to contain up to 360
BPF20
monomers per particle.
Example 5: Production of Eastern Equine Encephalitis virus (EEE) antigens in
CCMV VLPs in Pseudofnouas
5 A Synthesis of EEE Peptide Inserts
Two different EEE peptides (EEE-1 and EEE-2) were independently
expressed in CCMV VLPs.
EEE-peptide sequence:
DLDTHFTQYKLARPYIADCPNCGHS (SEQ ID N0:25)
EEE-1 nucleic acid sequence:
5'gacctggacacccacttcacccagtacaagctggcccgcccgtacatcgccgactgcccgaactgcggccacagc-
3' (SEQ ID N0:26)
EEE-2 peptide sequence:
GRLPRGEGDTFKGKLHVPFVPVKAK (SEQ ID N0:27)
EEE-2 nucleic acid sequence:
5'ggccgcctgccgcgcggcgaaggcgacaccttcaagggcaagctgcacgtgccgttcgtgccggtgaaggccaag-
3' (SEQ ID N0:28)
Nucleic acids encoding EEE-1 and EEE-2 were synthesized by SOE of
synthetic oligonucleotides. The resulting nucleic acids contained BamHI
recognition
site termini. The sense and anti-sense oligonucleotide primers for synthesis
of the
inserts included the BanzHI restriction sites and were as follows:
EEE1.S:
5' - cgg gga tcc tgg acc tgg aca ccc act tca ccc agt aca agc tgg ccc gcc cgt
ac - 3' (SEQ
ID N0:29)
EEE 1.AS
5' - cgc agg atc ccg ctg tgg ccg cag ttc ggg cag tcg gcg atg tac ggg cgg gcc
agc - 3'
(SEQ ID N0:30)
EEE2.S:
5' - cgg gga tcc tgg gcc gcc tgc cgc gcg gcg aag gcg aca cct tca agg gca agc -
3' (SEQ
ID N0:31)
EEE2.AS:
5' - cgc agg atc ccc ttg gcc ttc acc ggc acg aac ggc acg tgc agc ttg ccc ttg -
3' (SEQ ID
N0:32)
76


CA 02547511 2006-05-29
WO 2005/067478 PCT/US2004/040117
The resulting nucleic acids were digested with BamHI to create adhesive ends
for cloning into the pESC-CCMV129BamHI shuttle plasmid.
Each of the resulting EEE inserts was cloned in the pESC-CCMV129BamHI
shuttle plasmid at the BarnHI site of the CCMV129 CDS. Each resulting shuttle
plasmid was digested with SpeI and XhoI restriction enzymes. Each of the
desired
chimeric CCMV-129-EEE-encoding fragments was isolated by gel purification.
5 B Expression Plasmid Construction
The resulting chimeric CCMV129-EEE polynucleotide fragments were each
then inserted into the pMYC 1 X03 expression plasmid restricted with SpeI and
XhoI in
place of the buibui coding sequence, in operable attachment to the tac
promoter. The
resulting expression plasmid was screened by restriction digest with SpeI and
~aoI for
presence of the insert. The same protocols as described above for Example 1.B.
were
utilized.
S.C. Transformation and Expression
The resulting expression plasmid is transformed into P. fluofrescens MB214,
using protocols described above in Example 1.C. The same protocols as
described
above for Example 1.D. are used for expression of chimeric VLPs displaying the
EEE
antigens.
5 D Protein and VLP Recovery and Analysis
Shake-flask-cultured cells are lysed and fractionated, following the
procedures
of Example 1.E. The resulting fractions are analyzed by SDS-PAGE and Western
blotting as described for Example 1.F.
77

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Administrative Status

Title Date
Forecasted Issue Date Unavailable
(86) PCT Filing Date 2004-12-01
(87) PCT Publication Date 2005-07-28
(85) National Entry 2006-05-29
Examination Requested 2009-11-18
Dead Application 2011-12-01

Abandonment History

Abandonment Date Reason Reinstatement Date
2010-12-01 FAILURE TO PAY APPLICATION MAINTENANCE FEE

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Registration of a document - section 124 $100.00 2006-05-29
Application Fee $400.00 2006-05-29
Maintenance Fee - Application - New Act 2 2006-12-01 $100.00 2006-10-03
Maintenance Fee - Application - New Act 3 2007-12-03 $100.00 2007-09-10
Maintenance Fee - Application - New Act 4 2008-12-01 $100.00 2008-09-12
Request for Examination $800.00 2009-11-18
Maintenance Fee - Application - New Act 5 2009-12-01 $200.00 2009-12-01
Registration of a document - section 124 $100.00 2010-03-04
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
PFENEX INC.
Past Owners on Record
DAO, PHILIP PHUOC
DOW GLOBAL TECHNOLGIES INC.
RASOCHOVA, LADA
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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